Therapeutic methods using anti-CD200 antibodies

ABSTRACT

The present disclosure relates to anti-CD200 antibodies and to use of the antibodies in methods for treating autoimmune disorders and cancer. Also featured are biomarkers for use in selecting or prescribing a treatment modality for a patient with an autoimmune disorder and/or cancer. In addition, the disclosure features methods of treatment using an anti-CD200 antibody in combination with one or more additional therapeutic agents such as an anti-CD20 therapeutic agent.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a national stage filing under 35 U.S.C. §371 ofInternational Application No. PCT/US2011/024511, filed Feb. 11, 2011,which claims the benefit of the filing date under 35 U.S.C. §119(e) toU.S. provisional patent application Ser. No. 61/337,962, filed on Feb.11, 2010, the entire contents of which is hereby incorporated byreference. International Application No. PCT/US2011/024511 was publishedunder PCT Article 21(2) in English.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted via EFS-Web and is hereby incorporated by reference in itsentirety. Said ASCII copy, created on Aug. 10, 2012, is namedALXN1520.txtALXN152301 Seq.txt, and is 19,633 bytes in size.

TECHNICAL FIELD

The field of the invention is medicine, immunology, molecular biology,and protein chemistry.

BACKGROUND

Human CD200 protein is a type 1a transmembrane glycoprotein that isnormally expressed on thymocytes (e.g., T cells and B cells), neurons,and endothelial cells. Through engagement with its cognate receptor,CD200R, CD200 protein transduces an immunoregulatory signal that cansuppress T-cell-mediated immune responses. CD200 knockout animal studiesas well as experiments using antagonist anti-CD200 antibodies andrecombinant CD200-Fc fusion proteins have demonstrated that CD200protein is an immunosuppressive agent in autoimmune disorder and duringtransplantation. See, e.g., Hoek et al. (2000) Science 290:1768-1771 andGorczynski et al. (1999) J Immunol 163:1654-1660. The interactionbetween CD200 and CD200R results in altered cytokine profiles andpromotes a T_(H)2 T cell response over a T_(H)1 response. (See, e.g.,Kretz-Rommel (2007) J Immunol 178:5595-5605.)

SUMMARY

The present disclosure is based, at least in part, on the discovery bythe inventors that administration of an anti-CD200 antibody to an animalmodel of an autoimmune disease (autoimmune hemolytic disease) resultedin a marked decrease in production by the animal of disease-associatedautoantibodies. Administration of the anti-CD200 antibody also resultedin a marked delay in onset of production of autoantibodies in the mice.Because production of autoantibodies by a host is causative orassociated with a number of autoimmune disorders (e.g., myastheniagravis and Guillain-Barré syndrome), the inventors believe that ananti-CD200 antibody will be useful for treating patients suffering fromany one of a variety of autoimmune disorders.

Accordingly, in one aspect, the disclosure features a method fortreating an autoimmune disorder in a human. The method includesadministering to a human having an autoimmune disorder an amount of ananti-CD200 antibody that is sufficient to reduce in the human theconcentration of an autoantibody (or the production or expression of anautoantibody) associated with the autoimmune disorder to thereby treatthe autoimmune disorder.

In some embodiments, administration of the anti-CD200 antibody canreduce the autoantibody concentration in the blood of the human by atleast 10%, 20%, 50%, 75%, or more than 75%. In some embodiments,administration of the anti-CD200 antibody to the human can completelyeliminate detectable autoantibody in the human.

The disclosure also features a methods for preventing an autoimmunedisorder or delaying the onset of the autoimmune disorder, which methodincludes administering to a human having an autoimmune disorder anamount of an anti-CD200 antibody that is sufficient to: (i) prevent thegeneration, production, or expression by the human of an autoantibodyassociated with the autoimmune disorder or (ii) delay the generation of,or the onset of production or expression by the human of, theautoantibody associated with the autoimmune disorder, to thereby preventor delay the onset of the autoimmune disorder.

In yet another aspect, the disclosure features a method for treating anautoimmune disorder in a human, which method includes chronicallyadministering to a human having an autoimmune disorder an anti-CD200antibody in an amount and with a frequency sufficient to maintain in thehuman a reduced concentration of an autoantibody associated with theautoimmune disorder to thereby treat the autoimmune disorder.

In some embodiments, the anti-CD200 antibody can be administered to thehuman in an amount and with a frequency to maintain a greater than 10%,20%, 50%, 75%, or greater than 75% reduction in the concentration of theautoimmune antibody as compared to the concentration of the antibodyprior to administration of the anti-CD200 antibody.

The inventors also discovered several biomarkers evidencing theoccurrence in a human of an immunomodulatory effect by an anti-CD200antibody administered to animals with an autoimmune disorder. Forexample, the inventors have observed that following administration of ananti-CD200 antibody to an animal, the concentration of several leukocyteand bone marrow cell subsets is reduced in the animals. The inventorshave also discovered that the concentration of, e.g., F4/80⁺ lymphocytesin spleen are increased following administration of the anti-CD200antibody to the animal. While the disclosure is not bound by anyparticular theory or mechanism of action, the inventors believe thatmonitoring a patient treated with an anti-CD200 antibody for theoccurrence of one or more of these biomarkers is useful for, at bottom,determining whether the anti-CD200 antibody is capable of producing animmunomodulatory effect in the human to which the antibody isadministered. Moreover, one or more of the biomarkers are also usefulfor identifying a dose—a threshold dose—of an anti-CD200 antibody, suchas samalizumab (ALXN6000), that by virtue of its immunomodulatory effectin the human is sufficient to achieve a clinically-meaningful effect onthe disease (i.e., sufficient to treat a disease such as cancer or anautoimmune disorder). To wit, as described in the working examples ananti-CD200 antibody was capable of reducing the expression of autoimmuneantibodies in a mouse model of autoimmune disease.

Accordingly, the disclosure also features a method for treating adisorder in a human, the method comprising administering to a human inneed thereof an anti-CD200 antibody in an amount and with a frequencysufficient to treat the disorder by maintaining one or more of thefollowing physiological conditions in the human: (i) a decreased(reduced) concentration of at least one CD200⁺ leukocyte subset ascompared to a control concentration; (ii) an increased concentration ofF4/80⁺ cells as compared to a control concentration; and (iii) adecreased (reduced) concentration of at least one bone marrow stem cellsubset as compared to a control concentration. The disorder can be anydisorder that a medical practitioner reasonably believes can be treatedby a therapeutic anti-CD200 antibody. Such diseases include, e.g., acancer or an autoimmune disease.

In some embodiments, the at least one CD200⁺ leukocyte subset can be oneselected from the group consisting of CD3⁺/CD200⁺ cells, CD45R⁺/CD200⁺cells, CD5⁺/CD200⁺ cells, CD19⁺/CD200⁺ cells, CD138⁺/CD200⁺ cells, andCD200R⁺/CD200⁺ cells. In some embodiments, the at least one bone marrowstem cell subset can be one selected from the group consisting of CD200⁺bone marrow cells, Igk⁺/CD200⁺ bone marrow cells, CD138⁺/CD200⁺ bonemarrow cells, c-kit⁺/CD200⁺ bone marrow cells, andc-kit⁺/CD200⁺/Lin^(−/low) bone marrow cells. In some embodiments, theF4/80⁺ cells can be F4/80⁺ macrophages.

In some embodiments, at least one CD200⁺ leukocyte subset or the F4/80⁺cells can be present in the peripheral blood of the human. In someembodiments, the leukocytes or cells are resident in the spleen.

In some embodiments, the antibody can be administered to the human in anamount and with a frequency to maintain at least two, or all three, ofthe physiological conditions in the human.

In some embodiments, the autoimmune disorder can be a hemolytic disorderor an autoimmune hemolytic anemia (AIHA) such as any of the AIHA knownin the art of medicine (see below). In some embodiments, the autoimmunedisorder can be one selected from the group consisting of chronicobstructive pulmonary disease, diabetes mellitus type 1, Goodpasture'ssyndrome, Grave's disease, Guillain-Barré syndrome, IgA nephropathy,scleroderma, Sjögren's syndrome, Wegener's granulomatosis, pemphigusvulgaris, rheumatoid arthritis, cold agglutinin disease,anti-phospholipid syndrome, warm autoimmune hemolytic anemia, paroxysmalcold hemoglobinuria, Hashimoto's disease, idiopathic thrombocytopenicpurpura, myasthenia gravis, pulmonary biliary cirrhosis, and MillerFisher syndrome.

In some embodiments, the autoimmune disorder can be the result of, orcan be associated with, a cancer in the human. The cancer can be aliquid tumor such as, but not limited to, chronic lymphocytic leukemia(e.g., B cell chronic lymphocytic leukemia) or multiple myeloma.

In some embodiments, the methods described herein can includeadministering to the human at least one additional therapeutic agent fortreating an autoimmune disorder or a cancer.

The inventors have discovered that administration of an anti-CD200antibody to an animal resulted in a marked reduction in theconcentration of CD5⁺ cells (e.g., CD5⁺ leukocytes) in the spleen of theanimal. CD5 is a 67 kDa transmembrane glycoprotein that is expressed byT cells and a subset of B cells referred to as “B1 cells.” See, e.g.,Holodick et al. (2009) Eur J Immunol 39(9):2383-2394. B1 cells areintegrally involved in host defense against infections and CD5⁺ B1 cellsspontaneously and constitutively express immunoglobulin. Id. CD5expression by CLL cells has also been detected. In 1992, Almasri et al.reported that CD5⁺ CLL cells express lower levels of CD20 as determinedby flow cytometry. Am J Hematol 40:259, 261. See also Marti et al.(1992) “CD20 and CD5 expression in B-chronic lymphocytic leukemia” AnnN.Y. Acad Sci 651:480-483.

Rituximab is a chimeric, monoclonal anti-CD20 antibodyclinically-approved for the treatment of, among other things, chroniclymphocytic leukemia (CLL). See, e.g., Christian and Lin (2008) SeminHematol 45(2):95-103. Rituximab has been effective for treating CLL bothas a single agent and in combination, e.g., with the CHOP regimen(cyclophosphamide, doxorubicin, vincristine, and prednisone). Id.However, Ennishi et al. reported a correlation between CD5 expression byCLL cells and poor outcome in CLL patients treated with a combined RCHOPregimen (rituximab and CHOP regimen). (2008) Annals of Oncology 19:1921,1924 (FIG. 1). The report suggests that there exists a population ofpatients who receive less benefit from rituximab therapy and may requirealternative therapies.

While the disclosure is not bound by any particular theory or mechanismof action, it is likely that CD5⁺ CLL cells may be refractory torituximab therapy at least in part because of a reduced expression ofCD20. The inventors have shown that a therapeutic composition containingan anti-CD200 antibody is useful for reducing CD5⁺ cell populations inan animal and thus believe that the composition is particularly usefulfor treating a subset of CLL patients that are refractory to treatmentwith anti-CD20 therapy (e.g., rituximab-resistant).

Accordingly, the disclosure also features a method for treating a humanafflicted with a cancer or an autoimmune disorder, the method comprisingadministering to a human afflicted with a cancer or an autoimmunedisorder an anti-CD200 antibody in an amount that is sufficient to treatthe cancer or the autoimmune disorder, wherein the cancer or autoimmunedisorder is resistant, or is suspected of being resistant, or is likelyto become resistant, to therapy with an anti-CD20 therapeutic agent.

In another aspect, the disclosure features a method for treating a humanafflicted with a cancer, the method comprising administering to a humanafflicted with a cancer an anti-CD200 antibody in an amount that issufficient to treat the cancer, wherein the cancer is resistant, issuspected of being resistant, or is likely to become resistant, totherapy with an anti-CD20 therapeutic agent.

In another aspect, the disclosure features another method for treating ahuman afflicted with a cancer, the method comprising: identifying ahuman as having a cancer that is resistant, or is suspected to beresistant, to treatment with an anti-CD20 therapeutic agent; andadministering to the human an anti-CD200 antibody in an amount that iseffective to treat the cancer.

In some embodiments, the cancer can comprise or consist of cancer cellsthat express CD5.

In some embodiments, more than one dose (e.g., at least two, three,four, five, six, seven, eight, nine, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, or 20 or more doses) of the anti-CD200 antibody is administered tothe human. In some embodiments, more than 10 (e.g., more than 15, 20,25, 30, or 35 or more) doses of the anti-CD200 antibody are administeredto the human.

In some embodiments, the cancer is a solid tumor. Solid tumors include,e.g., lung cancer, breast cancer, colon cancer, pancreatic cancer, renalcancer, stomach cancer, liver cancer, bone cancer, neural tissue cancer(e.g., neuroblastoma), melanoma, thyroid cancer, ovarian cancer,testicular cancer, prostate cancer, cervical cancer, vaginal cancer, andbladder cancer. In some embodiments, the cancer is a liquid tumor.Liquid tumors include, e.g., leukemias (e.g., chronic lymphocyticleukemia such as B cell or T cell type chronic lymphocytic leukemia) andmultiple myeloma. Bone cancers include, without limitation, osteosarcomaand osteocarcinomas.

In yet another aspect, the disclosure features a method for treating ahuman afflicted with a liquid tumor. The method includes administeringto a human afflicted with a liquid tumor an anti-CD200 antibody in anamount that is sufficient to treat the liquid tumor, wherein at least aportion of the liquid tumor cells express CD5. The method can includedetermining whether the portion of liquid tumor cells express CD5.

In another aspect, the disclosure features a method for treating a humanafflicted with a liquid tumor, which method includes: identifying ahuman as having a liquid tumor comprising cells that express CD5; andadministering to the human an anti-CD200 antibody in an amount that issufficient to reduce the concentration of the CD5-expressing liquidtumor cells in the human to thereby treat the liquid tumor.

In another aspect, the disclosure features a method for treating a humanafflicted with a liquid tumor, the method comprising administering to ahuman afflicted with a liquid tumor an anti-CD200 antibody and ananti-CD20 therapeutic agent to thereby treat the liquid tumor, whereinat least a portion of the liquid tumor cells express CD5 prior toadministering the antibody and agent.

In another aspect, the disclosure features a method for treating a humanafflicted with a liquid tumor, wherein the method includes: identifyinga human as being afflicted with a liquid tumor comprising tumor cellsthat express CD5; and administering to the human an anti-CD200 antibodyand an anti-CD20 therapeutic agent to thereby treat the liquid tumor.

In some embodiments, the anti-CD20 therapeutic agent can be administeredprior to administration of the anti-CD200 antibody. In some embodiments,the anti-CD200 antibody is administered prior to administration of theanti-CD20 therapeutic agent. The anti-CD200 antibody and the anti-CD20therapeutic agent can be administered at the same time. The antibody canbe administered using the same administration route (e.g., intravenousadministration) or different route.

In some embodiments, the anti-CD200 antibody and anti-CD20 therapeuticagent can be administered to the human concurrently as a bispecificantibody that binds to human CD200 and to human CD20. That is, thetherapeutic agent administered to the human has both the properties ofan anti-CD200 antibody and the anti-CD20 therapeutic agent. In someembodiments, the bispecific anti-CD200 antibody/anti-CD20 antibody is aDVD-Ig antibody.

In some embodiments, at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, or 60% ormore of the liquid tumor cells express CD5.

The liquid tumor can be, e.g., a chronic lymphocytic leukemia ormultiple myeloma. The liquid tumor can be, e.g., a B cell chroniclymphocytic leukemia.

In some embodiments, the anti-CD20 therapeutic agent is an anti-CD20antibody such as, but not limited to, rituximab, ofatumumab, TRU-015,veltuzumab, ocrelizumab, or AME-133v.

In some embodiments, the anti-CD20 therapeutic agent is conjugated to atoxin. For example, in some embodiments, the anti-CD20 therapeutic agentis a toxin-antibody conjugate. The toxin can be, e.g., a small moleculedrug or a toxic polypeptide (e.g., ricin or saporin). In someembodiments, the toxin can be a bacterial toxin, a fungal toxin, or aplant toxin. In some embodiments, the toxin can be a radioactive agentsuch as, but not limited to, ⁹⁰Y, ¹⁸⁶Re, ¹⁸⁸Re, ⁶⁴Cu, ⁶⁷Cu, ²¹²Pb,²¹²Bi, ²¹³Bi, ¹²³I, ¹²⁵I, ¹³¹I, ¹¹¹In, ²¹¹At, ³²P, ¹⁷⁷Lu, ⁴⁷Sc, ¹⁰⁵Rh,¹⁰⁹Pd, ¹⁵³Sm, or ¹⁹⁹Au. In some embodiments, the toxin-antibodyconjugate is ⁹⁰Y-ibritumomab tiuxetan or ¹³¹I-tositumomab.

In some embodiments, the anti-CD200 antibody inhibits the interactionbetween CD200 and CD200R.

In some embodiments of any of the methods described herein, theanti-CD200 antibody can contains the following paired set of CDRs: aheavy chain CDR1 (HCDR1) comprising the amino acid sequence: GFTFSGFAMS(SEQ ID NO:4); a heavy chain CDR2 (HCDR2) comprising the amino acidsequence: SISSGGTTYYLDSVKG (SEQ ID NO:5); a heavy chain CDR3 (HCDR3)comprising the amino acid sequence: GNYYSGTSYDY (SEQ ID NO:6); a lightchain CDR1 (LCDR1) comprising the amino acid sequence: RASESVDSYGNSFMH(SEQ ID NO:7); a light chain CDR2 (LCDR2) comprising the amino acidsequence: RASNLES (SEQ ID NO:8); and a light chain CDR3 (LCDR3)comprising the amino acid sequence: QQSNEDPRT (SEQ ID NO:9).

In some embodiments of any of the methods described herein, theanti-CD200 antibody contains the following paired set of CDRs: a HCDR1comprising the amino acid sequence: GFNIKDYYMH (SEQ ID NO:10); a HCDR2comprising the amino acid sequence: WIDPENGDTKYAPKFQG (SEQ ID NO:11); aHCDR3 comprising the amino acid sequence: KNYYVSNYNFFDV (SEQ ID NO:12);a LCDR1 comprising the amino acid sequence: SASSSVRYMY (SEQ ID NO:13); aLCDR2 comprising the amino acid sequence: DTSKLAS (SEQ ID NO:14); and aLCDR3 comprising the amino acid sequence: FQGSGYPLT (SEQ ID NO:15).

In some embodiments of any of the methods described herein, theanti-CD200 antibody contains the following paired set of CDRs: a HCDR1comprising the amino acid sequence: GFNIKDYYIH (SEQ ID NO:16); a HCDR2comprising the amino acid sequence: WIDPEIGATKYVPKFQG (SEQ ID NO:17); aHCDR3 comprising the amino acid sequence: LYGNYDRYYAMDY (SEQ ID NO:18);a LCDR1 comprising the amino acid sequence: KASQNVRTAVA (SEQ ID NO:19);a LCDR2 comprising the amino acid sequence: LASNRHT (SEQ ID NO:20); anda LCDR3 comprising the amino acid sequence: LQHWNYPLT (SEQ ID NO:21).

In some embodiments of any of the methods described herein, theanti-CD200 antibody contains the following paired set of CDRs: a HCDR1comprising the amino acid sequence: GYSFTDYIIL (SEQ ID NO:22); a HCDR2comprising the amino acid sequence: HIDPYYGSSNYNLKFKG (SEQ ID NO:23); aHCDR3 comprising the amino acid sequence: SKRDYFDY (SEQ ID NO:24); aLCDR1 comprising the amino acid sequence: KASQDINSYLS (SEQ ID NO:25); aLCDR2 comprising the amino acid sequence: RANRLVD (SEQ ID NO:26); and aLCDR3 comprising the amino acid sequence: LQYDEFPYT (SEQ ID NO:27).

In some embodiments of any of the methods described herein, theanti-CD200 antibody contains the following paired set of CDRs: a HCDR1comprising the amino acid sequence: GYTFTEYTMH (SEQ ID NO:28); a HCDR2comprising the amino acid sequence: GVNPNNGGALYNQKFKG (SEQ ID NO:29); aHCDR3 comprising the amino acid sequence: RSNYRYDDAMDY (SEQ ID NO:30); aLCDR1 comprising the amino acid sequence: KSSQSLLDIDEKTYLN (SEQ IDNO:31); a LCDR2 comprising the amino acid sequence: LVSKLDS (SEQ IDNO:32); and a LCDR3 comprising the amino acid sequence: WQGTHFPQT (SEQID NO:33).

In some embodiments of any of the methods described herein, theanti-CD200 antibody contains the following paired set of CDRs: a HCDR1comprising the amino acid sequence: AFNIKDHYMH (SEQ ID NO:34); a HCDR2comprising the amino acid sequence: WIDPESGDTEYAPKFQG (SEQ ID NO:35); aHCDR3 comprising the amino acid sequence: FNGYQALDQ (SEQ ID NO:36); aLCDR1 comprising the amino acid sequence: TASSSVSSSYLH (SEQ ID NO:37); aLCDR2 comprising the amino acid sequence: STSNLAS (SEQ ID NO:38); and aLCDR3 comprising the amino acid sequence: RQYHRSPPIFT (SEQ ID NO:39).

In some embodiments, the anti-CD200 antibody and/or the anti-CD20antibody is an IgG1, IgG2, IgG2a, IgG3, IgG4, IgM, IgA1, IgA2, IgA, IgD,or IgE antibody. In some embodiments, the anti-CD200 antibody and/or theanti-CD20 antibody is a murine antibody, a chimeric antibody, ahumanized antibody, a single chain antibody, or a human antibody. Insome embodiments, the anti-CD200 antibody or the anti-CD20 antibody isan antigen-binding antibody fragment selected from the group consistingof a Fab fragment, a F(ab′)₂ fragment, a Fab′ fragment, an scFvfragment, a minibody, a diabody, or a triabody

In yet another aspect, the disclosure features a method for selecting atherapy for a patient afflicted with a liquid tumor, the methodcomprising: identifying a patient as having a liquid tumor comprisingtumor cells that express CD5; and selecting for the patient ananti-CD200 antibody for use in treating the liquid tumor.

In another aspect, the disclosure features a method for prescribing atherapy for a patient afflicted with a liquid tumor, the methodcomprising: identifying a patient as having a liquid tumor comprisingtumor cells that express CD5; and prescribing for the patient ananti-CD200 antibody for use in treating the liquid tumor. The anti-CD200antibody can be a bispecific antibody such as one that comprises a firstantigen-combining site and a second antigen-combining site, wherein thefirst antigen-combining site binds to CD200 and the secondantigen-combining site binds to CD20.

In yet another aspect, the disclosure features a bispecific antibodythat comprises a first antigen-combining site and a secondantigen-combining site, wherein the first antigen-combining site bindsto CD200 and the second antigen-combining site binds to CD20. Thebispecific antibody can be, e.g., an IgG1, IgG2, IgG2a, IgG3, IgG4, IgM,IgA1, IgA2, IgA, IgD, or IgE antibody. In some embodiments, thebispecific anti-CD200/anti-CD20 antibody is a murine antibody, achimeric antibody, a humanized antibody, a single chain antibody, or ahuman antibody. In some embodiments, the bispecific antibody can be usedin any of the methods described herein (e.g., treating cancer or anautoimmune disease).

In yet another aspect, the disclosure features: (i) a nucleic acidencoding the bispecific antibody; (ii) a vector (e.g., an expressionvector) comprising the nucleic acid; and (iii) a cell comprising thevector. In another aspect, the disclosure features a method forproducing the antibody, the method comprising culturing the cell for atime and under conditions sufficient to allow for production of theantibody in the cell. The method can also include the step of isolatingthe bispecific antibody from the cell or from the media in which thecell is cultured.

In some embodiments, the bispecific antibody is a single chain diabody,a tandem single chain Fv fragment, a tandem single chain diabody, or afusion protein comprising a single chain diabody and at least a portionof an immunoglobulin heavy chain constant region. In some embodiments,the bispecific antibody is a dual variable domain immunoglobulin.

In some embodiments, the first antigen combining site binds to a humanCD200 protein, e.g., a human CD200 protein comprising the amino acidsequence depicted in any one of SEQ ID NOs:1 to 3. In some embodiments,the second antigen combining site binds to a human CD20 protein, e.g., ahuman CD20 protein comprising the amino acid sequence depicted in anyone of SEQ ID NOs: 40 to 42. In some embodiments, the second antigencombining site binds to a human CD20 protein at an epitope thatcomprises at least part (e.g., at least 4 amino acids) of the amino acidsequence depicted in SEQ ID NO:41 and at least part (e.g., at least 4amino acids) of the amino acid sequence depicted in SEQ ID NO:42.

In some embodiments, the first antigen combining site is obtained fromsamalizumab. In some embodiments, the second antigen combining site isobtained from rituximab, of atumumab, TRU-015, veltuzumab, ocrelizumab,or AME-133v. The bispecific antibody can be conjugated to, or contain, aheterologous moiety such as a detectable label or a toxin.

“Polypeptide,” “peptide,” and “protein” are used interchangeably andmean any peptide-linked chain of amino acids, regardless of length orpost-translational modification. The CD200 proteins described herein cancontain or be wild-type proteins or can be variants that have not morethan 50 (e.g., not more than one, two, three, four, five, six, seven,eight, nine, ten, 12, 15, 20, 25, 30, 35, 40, or 50) conservative aminoacid substitutions. Conservative substitutions typically includesubstitutions within the following groups: glycine and alanine; valine,isoleucine, and leucine; aspartic acid and glutamic acid; asparagine,glutamine, serine and threonine; lysine, histidine and arginine; andphenylalanine and tyrosine.

The CD200 proteins and CD20 proteins described herein also include“antigenic peptide fragments” of the proteins, which are shorter thanfull-length proteins, but retain at least 10% (e.g., at least 10%, atleast 15%, at least 20%, at least 25%, at least 30%, at least 35%, atleast 40%, at least 50%, at least 55%, at least 60%, at least 70%, atleast 80%, at least 90%, at least 95%, at least 98%, at least 99%, atleast 99.5%, or 100% or more) of the ability of the full-length proteinto induce an antigenic response in a mammal (see below under “Methodsfor Producing an Antibody”). Antigenic peptide fragments of a CD200protein or a CD20 protein include terminal as well internal deletionvariants of the protein. Deletion variants can lack one, two, three,four, five, six, seven, eight, nine, ten, 11, 12, 13, 14, 15, 16, 17,18, 19, or 20 amino acid segments (of two or more amino acids) ornon-contiguous single amino acids. Antigenic peptide fragments can be atleast 6 (e.g., at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75,80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200or more) amino acid residues in length (e.g., at least 6 contiguousamino acid residues in any one of SEQ ID NOs:1 to 3). In someembodiments, an antigenic peptide fragment of a human CD200 protein isless than 225 (e.g., less than 200, 190, 180, 170, 160, 150, 140, 130,120, 110, 100, 95, 90, 85, 80, 75, 60, 50, 49, 48, 47, 46, 45, 44, 43,42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25,24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, or 7)amino acid residues in length (e.g., less than 225 contiguous amino acidresidues in any one of SEQ ID NOs:1 to 3). In some embodiments, anantigenic peptide fragment of a full-length CD200 protein is at least 6,but less than 225, amino acid residues in length.

In some embodiments, the human CD200 protein can have an amino acidsequence that is, or is greater than, 70 (e.g., 71, 72, 73, 74, 75, 76,77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,95, 96, 97, 98, 99, or 100) % identical to the human CD200 proteinhaving the amino acid sequence depicted in SEQ ID NO:1 or SEQ ID NO:2(see below). In some embodiments, a human CD20 protein can have an aminoacid sequence that is, or is greater than, 70 (e.g., 71, 72, 73, 74, 75,76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93,94, 95, 96, 97, 98, 99, or 100) % identical to the human CD200 proteinhaving the amino acid sequence depicted in SEQ ID NO:40.

Percent (%) amino acid sequence identity is defined as the percentage ofamino acids in a candidate sequence that are identical to the aminoacids in a reference sequence, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent sequenceidentity. Alignment for purposes of determining percent sequenceidentity can be achieved in various ways that are within the skill inthe art, for instance, using publicly available computer software suchas BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software.Appropriate parameters for measuring alignment, including any algorithmsneeded to achieve maximal alignment over the full-length of thesequences being compared can be determined by known methods.

Amino acid sequences for exemplary human CD200 proteins and human CD20proteins, as well as antigenic peptide fragments thereof are known inthe art and are set forth below.

As used herein, the term “antibody” refers to a whole or intact antibodymolecule (e.g., IgM, IgG (including IgG1, IgG2, IgG3, and IgG4), IgA,IgD, or IgE) or any antigen-binding fragment thereof. The term antibodyincludes, e.g., a chimerized or chimeric antibody, a humanized antibody,a deimmunized antibody, and a fully human antibody. Antigen-bindingfragments of an antibody include, e.g., a single chain antibody, asingle chain Fv fragment (scFv), an Fd fragment, an Fab fragment, anFab′ fragment, or an F(ab′)₂ fragment. An scFv fragment is a singlepolypeptide chain that includes both the heavy and light chain variableregions of the antibody from which the scFv is derived. In addition,intrabodies, minibodies, triabodies, and diabodies (see, e.g.,Todorovska et al. (2001) J Immunol Methods 248(1):47-66; Hudson andKortt (1999) J Immunol Methods 231(1):177-189; Poljak (1994) Structure2(12):1121-1123; Rondon and Marasco (1997) Annual Review of Microbiology51:257-283, the disclosures of each of which are incorporated herein byreference in their entirety) are also included in the definition ofantibody and are compatible for use in the methods described herein.Bispecific antibodies (including DVD-Ig antibodies; see below) are alsoembraced by the term “antibody.” Bispecific antibodies are monoclonal,preferably human or humanized, antibodies that have bindingspecificities for at least two different antigens.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure pertains. In case of conflict, thepresent document, including definitions, will control. Preferred methodsand materials are described below, although methods and materialssimilar or equivalent to those described herein can also be used in thepractice or testing of the presently disclosed methods and compositions.All publications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety.

Other features and advantages of the present disclosure, e.g., methodsfor treating a rituximab-resistant cancer (e.g., chronic lymphocyticleukemia), will be apparent from the following description, theexamples, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a line graph depicting the delay in anti-mouse RBCautoantibody production in mice with autoimmune hemolytic diseasetreated with an anti-CD200 antibody. The Y-axis represents the incidence(%) of autoantibody production in the mice in each group. The X-axisrepresents the time in which the presence of autoantibodies in eachmouse was detected. The seven groups of mice evaluated included: micethat were immunized with rat RBCs, but not treated with an antibody (NoRx); mice that were immunized with rat RBCs and treated with a controlantibody (Cntrl Ab); mice that were immunized with rat RBCs and treatedwith an anti-CD200 antibody (Antibody 1); mice that were immunized withrat RBCs and treated with cyclosporine (CsA); mice that were immunizedwith rat RBCs and treated with the control antibody and cyclosporine A(Cntrl Ab+CsA); mice that were immunized with rat RBCs and treated withan anti-CD200 antibody and cyclosporine A (Antibody 1+CsA); and micethat were neither immunized with rat RBCs nor treated with antibody orcyclosporine (No-imm No Rx).

FIG. 2 is a line graph depicting the effect of an anti-CD200 antibody onanti-RBC antibody titer in a mouse model of autoimmune hemolyticdisease. C57BL/6 mice were administered 2×10⁸ rat RBCs intraperitoneally(i.p.) once on study day 0 and then once per week thereafter for theremainder of the study. The rat RBC-immunized mice were then treatedwith an anti-CD200 antibody that possessed effector function (Antibody1; Ab 1) at 5 mg/kg or 1 mg/kg; an anti-CD200 antibody that did notpossess effector function (Antibody 2; Ab 2) at 5 mg/kg; or a controlantibody (Cntl) at 5 mg/kg. A group of mice was also treated withvehicle only. A final group of mice received no immunization or antibodytreatment (NC). The Y-axis depicts the relative fluorescence intensityreflected as the OD405× serum dilution factor and the X-axis representsthe number of days following the start of the study.

FIG. 3 is a bar graph depicting the reduction in antigen-inducedproliferation of splenocytes isolated from mice treated with ananti-CD200 antibody. The Y-axis represents the mean counts per minute of³H-thymidine radioactivity in nucleic acid isolated from each cellpopulation. The X-axis represents individual mice, three (3) depicted ineach group. For each mouse, the four measurements are for proliferationof splenocytes induced by medium alone, mouse red blood cells (mRBC),rat red blood cells (rRBC), or bovine serum albumin (BSA). The mice ofGroup 1 were treated with an anti-CD200 antibody with effector function(Antibody 1) at a dose of 5 mg/kg. The mice of Group 2 were treated withAntibody 1 at a dose of 1 mg/kg. The mice of Group 3 were treated with acontrol antibody that does not bind to CD200 and the mice of Group 4were not treated with an antibody or immunized with the rat red bloodcells.

FIG. 4 is a bar graph depicting the reduction in CD200⁺ splenocytes inmice treated with an anti-CD200 antibody. C57BL/6 mice were administered2×10⁸ rat RBCs intraperitoneally (i.p.) once on study day 0 and thenonce per week thereafter for the remainder of the study. The ratRBC-immunized mice were then treated with an anti-CD200 antibody thatpossessed effector function (Antibody 1; Ab 1) at 5 mg/kg or 1 mg/kg; ananti-CD200 antibody that did not possess effector function (Antibody 2;Ab 2) at 5 mg/kg; or a control antibody (Cntl) at 5 mg/kg. A group ofmice was also treated with vehicle only. A final group of mice receivedno immunization or antibody treatment (Un-imm, No-Ab). The Y-axisrepresents the percentage of CD200⁺ cells in the total population ofviable splenocytes. The X-axis represents individual mice, three (3)depicted in each group.

DETAILED DESCRIPTION

The present disclosure relates to anti-CD200 antibodies and to use ofthe antibodies in methods for treating autoimmune disorders or cancer.Also featured are biomarkers for use in selecting or prescribing atreatment modality for a patient with an autoimmune disorder and/orcancer. In addition, the disclosure features methods of treatment usingan anti-CD200 antibody in combination with one or more additionaltherapeutic agents such as an anti-CD20 therapeutic agent. While in noway intended to be limiting, exemplary anti-CD200 antibodies andCD200-binding fragments thereof, conjugates, pharmaceutical compositionsand formulations, biomarkers, and methods employing any of the foregoingare elaborated on below and are exemplified in the working Examples.

Anti-CD200 Antibodies

The disclosure features antibodies that bind to a human CD200polypeptide (sometimes the antibodies are referred to herein as“anti-CD200 antibodies”). Also featured are antigen-binding(CD200-binding) fragments of the antibodies. In some embodiments, ananti-CD200 antibody described herein binds to an epitope in the humanCD200 protein. For example, the anti-CD200 antibody can bind to anepitope in the human CD200 protein comprising, or consisting of, thefollowing amino acid sequence:MERLVIRMPFSHLSTYSLVWVMAAVVLCTAQVQVVTQDEREQLYTPASLKCSLQNAQEALIVTWQKKKAVSPENMVTFSENHGVVIQPAYKDKINITQLGLQNSTITFWNITLEDEGCYMCLFNTFGFGKISGTACLTVYVQPIVSLHYKFSEDHLNITCSATARPAPMVFWKVPRSGIENSTVTLSHPNGTTSVTSILHIKDPKNQVGKEVICQVLHLGTVTDFKQTVNKGYWFSVPLLLSIVSLVILLVLISILLYWKRHRNQ DREP (SEQ IDNO:1; Genbank Accession No. NP_(—)005935.2). SEQ ID NO:1 depicts theamino acid sequence for a full-length, precursor human CD200 isoform Aprotein. In some embodiments, an anti-CD200 antibody described hereinbinds to an epitope in the human CD200 protein comprising, or consistingof, the following amino acid sequence:MERLTLTRTIGGPLLTATLLGKTTINDYQVIRMPFSHLSTYSLVWVMAAVVLCTAQVQVVTQDEREQLYTPASLKCSLQNAQEALIVTWQKKKAVSPENMVTFSENHGVVIQPAYKDKINITQLGLQNSTITFWNITLEDEGCYMCLFNTFGFGKISGTACLTVYVQPIVSLHYKFSEDHLNITCSATARPAPMVFWKVPRSGIENSTVTLSHPNGTTSVTSILHIKDPKNQVGKEVICQVLHLGTVTDFKQTVNKGYWFSVPLLLSIVSLVILLVLISILLYWKRHRNQDREP (SEQ ID NO:2; Genbank Accession No.NP_(—)001004196.2). SEQ ID NO:2 depicts the amino acid sequence of afull-length CD200 isoform B protein. In some embodiments, the anti-CD200antibody binds to an epitope present in a human CD200 protein having thefollowing amino acid sequence:VIRMPFSHLSTYSLVWVMAAVVLCTAQVQVVTQDEREQLYTTASLKCSLQNAQEALIVTWQKKKAVSPENMVTFSENHGVVIQPAYKDKINITQLGLQNSTITFWNITLEDEGCYMCLFNTFGFGKISGTACLTVYVQPIVSLHYKFSEDHLNITCSATARPAPMVFWKVPRSGIENSTVTLSHPNGTTSVTSILHIKDPKNQVGKEVICQVLHLGTVTDFKQTVNKGYWFSVPLLLSIVSLVILLVLISILLYWKRHRNQDR GELSQGVQKMT

(SEQ ID NO:3; Genbank Accession No. CAA28943.1; FIG. 3 of McCaughan etal. (1987) Immunogenetics 25:329-335). SEQ ID NO:3 is an exemplarysequence for a full-length human CD200 protein.

In some embodiments, an anti-CD200 antibody described herein binds to anepitope within the extracellular portion of a CD200 protein. Forexample, in some embodiments, the anti-CD200 antibody can bind to CD200protein at an epitope within or overlapping with: (i) amino acids 1 to233 of the amino acid sequence depicted in SEQ ID NO:1; (ii) amino acids1 to 258 of the amino acid sequence depicted in SEQ ID NO:2; or aminoacids 1 to 229 of the amino acid sequence depicted in SEQ ID NO:3.

In some embodiments, the anti-CD200 antibody binds to an epitope in thehuman CD200 protein lacking the leader sequence. For example, ananti-CD200 antibody described herein can bind to a CD200 protein at anepitope within or overlapping with amino acids 31 to 233 of the aminoacid sequence depicted in SEQ ID NO:1, which corresponds to theextracellular portion of the mature form of human CD200 isoform A lessthe amino terminal leader sequence. In some embodiments, an anti-CD200antibody described herein can bind to a CD200 protein at an epitopewithin or overlapping with amino acids 56 to 258 of the amino acidsequence depicted in SEQ ID NO:2, which corresponds to the extracellularportion of the mature form of human CD200 isoform B less the aminoterminal leader sequence. In some embodiments, an anti-CD200 antibodydescribed herein can bind to a CD200 protein at an epitope within oroverlapping with amino acids 27 to 229 of the amino acid sequencedepicted in SEQ ID NO:3, which corresponds to the extracellular portionof the mature form of human CD200 less the amino terminal leadersequence.

An “epitope” refers to the site on a protein (e.g., a human CD200protein) that is bound by an antibody. “Overlapping epitopes” include atleast one (e.g., two, three, four, five, or six) common amino acidresidue(s).

In some embodiments, the anti-CD200 antibody specifically binds to ahuman CD200 protein (e.g., the human CD200 protein having the amino acidsequence depicted in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or theextracellular domains of the mature forms of the CD200 proteins). Theterms “specific binding” or “specifically binds” refer to two moleculesforming a complex (e.g., a complex between an anti-CD200 antibody and aCD200 protein) that is relatively stable under physiologic conditions.Typically, binding is considered specific when the association constant(K_(a)) is higher than 10⁶ M⁻¹. Thus, an anti-CD200 antibody canspecifically bind to a CD200 protein with a K_(a) of at least (orgreater than) 10⁶ (e.g., at least or greater than 10⁷, 10⁸, 10⁹, 10¹⁰,10¹¹10¹², 10¹³, 10¹⁴, or 10¹⁵ or higher) M⁻¹. Examples of antibodiesthat specifically bind to a human CD200 protein are described in, e.g.,U.S. Pat. Nos. 7,408,041; 7,427,665; 7,435,412; and 7,598,353, thedisclosures of each of which are incorporated herein by reference intheir entirety.

The amino acid sequences for several exemplary anti-CD200 antibodies aredescribed in, e.g., U.S. Pat. No. 7,408,041. For example, the anti-CD200antibody can comprise the amino acid sequence of the heavy and lightchain variable regions of one of the Fab antibodies—d1B10, d1A5, d1B5,c2aB7, c1A10, or c2aA10—depicted in FIG. 23 of U.S. Pat. No. 7,408,041,the sequences depicted in FIG. 23 being incorporated herein by referencein their entirety. In some embodiments, an anti-CD200 antibody describedherein contains a paired set of heavy chain CDRs and light chain CDRs ofone of the Fab antibodies depicted in FIG. 23 of U.S. Pat. No.7,408,041. For example, an anti-CD200 antibody described herein containsthe paired set of CDRs from the d1B10 Fab antibody: a heavy chain CDR1(HCDR1) comprising the following sequence: GFTFSGFAMS (SEQ ID NO:4); aheavy chain CDR2 (HCDR2) comprising the following sequence:SISSGGTTYYLDSVKG (SEQ ID NO:5); a heavy chain CDR3 (HCDR3) comprisingthe following sequence: GNYYSGTSYDY (SEQ ID NO:6); a light chain CDR1(LCDR1) comprising the following sequence: RASESVDSYGNSFMH (SEQ IDNO:7); a light chain CDR2 (LCDR2) comprising the following sequence:RASNLES (SEQ ID NO:8); and a light chain CDR3 (LCDR3) comprising thefollowing sequence: QQSNEDPRT (SEQ ID NO:9).

In another example, an anti-CD200 antibody described herein can containthe paired set of CDRs from the d1A5 Fab antibody: (i) a HCDR1comprising the following sequence: GFNIKDYYMH (SEQ ID NO:10); a HCDR2comprising the following sequence: WIDPENGDTKYAPKFQG (SEQ ID NO:11); aHCDR3 comprising the following sequence: KNYYVSNYNFFDV (SEQ ID NO:12); aLCDR1 comprising the following sequence: SASSSVRYMY (SEQ ID NO:13); aLCDR2 comprising the following sequence: DTSKLAS (SEQ ID NO:14); and aLCDR3 comprising the following sequence: FQGSGYPLT (SEQ ID NO:15).

In another example, an anti-CD200 antibody described herein can comprisethe paired set of CDRs from the d1B5 Fab antibody: a HCDR1 comprisingthe following amino acid sequence: GFNIKDYYIH (SEQ ID NO:16); a HCDR2comprising the following amino acid sequence: WIDPEIGATKYVPKFQG (SEQ IDNO:17); a HCDR3 comprising the following amino acid sequence:LYGNYDRYYAMDY (SEQ ID NO:18); a LCDR1 comprising the following aminoacid sequence: KASQNVRTAVA (SEQ ID NO:19); a LCDR2 comprising thefollowing amino acid sequence: LASNRHT (SEQ ID NO:20); and a LCDR3comprising the following amino acid sequence: LQHWNYPLT (SEQ ID NO:21).

In another example, an anti-CD200 antibody described herein can containthe paired set of CDRs from the c2aB7 Fab antibody: a HCDR1 comprisingthe amino acid sequence: GYSFTDYIIL (SEQ ID NO:22); a HCDR2 comprisingthe amino acid sequence: HIDPYYGSSNYNLKFKG (SEQ ID NO:23); a HCDR3comprising the amino acid sequence: SKRDYFDY (SEQ ID NO:24); a LCDR1comprising the amino acid sequence: KASQDINSYLS (SEQ ID NO:25); a LCDR2comprising the amino acid sequence: RANRLVD (SEQ ID NO:26); and a LCDR3comprising the amino acid sequence: LQYDEFPYT (SEQ ID NO:27).Samalizumab (ALXN6000) contains the aforementioned paired CDR set of thec2aB7 Fab antibody originally set forth in FIG. 23 of U.S. Pat. No.7,408,041.

In yet another example, an anti-CD200 antibody described herein cancontain a paired set of CDRs from the c1A10 Fab antibody: a HCDR1comprising the amino acid sequence: GYTFTEYTMH (SEQ ID NO:28); a HCDR2comprising the amino acid sequence: GVNPNNGGALYNQKFKG (SEQ ID NO:29); aHCDR3 comprising the amino acid sequence: RSNYRYDDAMDY (SEQ ID NO:30); aLCDR1 comprising the amino acid sequence: KSSQSLLDIDEKTYLN (SEQ IDNO:31); a LCDR2 comprising the amino acid sequence: LVSKLDS (SEQ IDNO:32); and a LCDR3 comprising the amino acid sequence: WQGTHFPQT (SEQID NO:33).

And in yet another example, an anti-CD200 antibody described herein cancontain a paired set of CDRs from the c2aA10 Fab antibody: a HCDR1comprising the amino acid sequence: AFNIKDHYMH (SEQ ID NO:34); a HCDR2comprising the amino acid sequence: WIDPESGDTEYAPKFQG (SEQ ID NO:35); aHCDR3 comprising the amino acid sequence: FNGYQALDQ (SEQ ID NO:36); aLCDR1 comprising the amino acid sequence: TASSSVSSSYLH (SEQ ID NO:37); aLCDR2 comprising the amino acid sequence: STSNLAS (SEQ ID NO:38); and aLCDR3 comprising the amino acid sequence: RQYHRSPPIFT (SEQ ID NO:39).

Additional exemplary sets of CDRs of anti-CD200 antibodies are describedin, e.g., U.S. Pat. No. 7,427,665. In some embodiments, the anti-CD200antibody is samalizumab (ALXN6000).

Methods for determining whether an antibody binds to a protein antigenand/or the affinity for an antibody to a protein antigen are known inthe art. For example, the binding of an antibody to a protein antigencan be detected and/or quantified using a variety of techniques such as,but not limited to, Western blot, dot blot, surface plasmon resonancemethod (e.g., BIAcore system; Pharmacia Biosensor AB, Uppsala, Swedenand Piscataway, N.J.), or enzyme-linked immunosorbent assay (ELISA).See, e.g., Harlow and Lane (1988) “Antibodies: A Laboratory Manual” ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Benny K. C. Lo(2004) “Antibody Engineering: Methods and Protocols,” Humana Press(ISBN: 1588290921); Borrebaek (1992) “Antibody Engineering, A PracticalGuide,” W.H. Freeman and Co., NY; Borrebaek (1995) “AntibodyEngineering,” 2^(nd) Edition, Oxford University Press, NY, Oxford; Johneet al. (1993) J Immunol Meth 160:191-198; Jonsson et al. (1993) Ann BiolClin 51:19-26; and Jonsson et al. (1991) Biotechniques 11:620-627.

In some embodiments, the anti-CD200 antibody can crossblock binding ofanother antibody that binds to an epitope within, or overlapping with, ahuman CD200 protein. In some embodiments, the anti-CD200 antibody cancrossblock binding of an antibody that binds to an epitope within, oroverlapping with, a peptide fragment of a human CD200 protein. Thepeptide fragment can be a fragment of a human CD200 protein having theamino acid sequence depicted in, e.g., any one of SEQ ID NOs:1 to 3. Asused herein, the term “crossblocking antibody” refers to an antibodythat lowers the amount of binding of anti-CD200 antibody to an epitopeon a CD200 protein relative to the amount of binding of the anti-CD200antibody to the epitope in the absence of the antibody. Suitable methodsfor determining whether a first antibody crossblocks binding of a secondantibody to an epitope are known in the art.

Methods for identifying the epitope to which a particular antibody(e.g., an anti-CD200 antibody) binds are also known in the art. Forexample, the binding epitope of an anti-CD200 antibody can be identifiedby measuring the binding of the antibody to several (e.g., three, four,five, six, seven, eight, nine, 10, 15, 20, or 30 or more) overlappingpeptide fragments of a CD200 protein (e.g., several overlappingfragments of a protein having the amino acid sequence depicted in, e.g.,any one of SEQ ID NOs:1 to 3). Each of the different overlappingpeptides is then bound to a unique address on a solid support, e.g.,separate wells of a multi-well assay plate. Next, the anti-CD200antibody is interrogated by contacting it to each of the peptides in theassay plate for an amount of time and under conditions that allow forthe antibody to bind to its epitope. Unbound anti-CD200 antibody isremoved by washing each of the wells. Next, a detectably-labeledsecondary antibody that binds to the anti-CD200 antibody, if present ina well of the plate, is contacted to each of the wells, and unboundsecondary antibody is removed by washing steps. The presence or amountof the detectable signal produced by the detectably-labeled secondaryantibody in a well is an indication that the anti-CD200 antibody bindsto the particular peptide fragment associated with the well. See, e.g.,Harlow and Lane (supra), Benny K. C. Lo (supra), and U.S. PatentApplication Publication No. 20060153836, the disclosure of which isincorporated by reference in its entirety. A particular epitope to whichan antibody binds can also be identified using BIAcore chromatographictechniques (see, e.g., Pharmacia BIAtechnology Handbook, “EpitopeMapping,” Section 6.3.2, (May 1994); and Johne et al. (1993) J ImmunolMethods 160:20191-8).

In some embodiments, an anti-CD200 antibody, or a CD200-binding fragmentthereof, described herein binds to a human CD200 polypeptide expressedon the surface of a cell. Methods for determining whether an antibodybinds to a protein expressed on the surface of a cell are known in theart and described in, e.g., Petermann et al. (2007) J Clin Invest117(12):3922-9; Rijkers et al. (2008) Mol Immunol 45(4):1126-35; andKretz-Rommel (2007) J Immunol 178(9):5595-605.

In some embodiments, an anti-CD200 antibody or CD200-binding fragmentthereof described herein inhibits the interaction between CD200 proteinand the CD200 receptor. Methods for determining whether an agent (suchas an antibody) inhibits the interaction between CD200 and CD200R areknown in the art and described in, e.g., Hatherly and Barclay (2004) EurJ Immunol 34(6):1688-94.

In some embodiments, the anti-CD200 antibody or CD200-binding fragmentthereof inhibits the formation of osteoclasts in vitro and/or in vivo.Suitable methods for determining whether an antibody inhibits theformation of osteoclasts are known in the art and described in, e.g.,PCT Publication No. WO 2008/089022 and Cui et al. (2007) Proc Natl AcadSci USA 104(36):14436-14441. For example, murine bone marrow cells canbe cultured in the presence of, e.g., RANKL and M-CSF in the presence orabsence of an anti-CD200 antibody. A decrease in the percentage ofosteoclasts formed from the bone marrow cells in the presence of theantibody as compared to the percentage of osteoclasts formed in theabsence of the antibody indicates that the antibody inhibits osteoclastformation in vitro.

Since CD200 is expressed on normal cells such as endothelial cells,albeit at lower levels than on cancer cells, it could be in someembodiments advantageous to administer a variant anti-CD200 antibody (orCD200-binding fragment thereof) with a constant region modified so thatit does not mediate, or has decreased ability to mediate,antibody-dependent cell-mediated cytotoxicity (ADCC), whereby antibodiesbind Fc receptors on natural killer (NK) cells or macrophages leading tocell death, or complement-dependent cytotoxicity (CDC), which is celldeath induced via activation of the complement cascade (reviewed inDaeron (1997) Annu Rev Immunol 15:203-234; Ward and Ghetie (1995)Therapeutic Immunol 2:77-94; and Ravetch and Kinet (1991) Annu RevImmunol. 9:457-492). Such a modification would be useful to limit damageto normal cells. CD200 expression is also upregulated on some activatednormal cells (e.g., activated T cells), rendering such cells vulnerableto killing by an anti-CD200 antibody with effector function. It may beadvantageous to use an anti-CD200 antibody lacking effector function toavoid killing of these cells by ADCC or CDC. The effector function of ananti-CD200 antibody can be eliminated by replacing an immunoglobulinconstant region that has effector function (e.g., the IgG1 constantdomain) for a constant region that does not have effector function(e.g., an IgG2/IgG4 fusion constant region). See, e.g., Burton et al.(1992) Adv Immun 51:1-18; Canfield et al. (1991) J Exp Med173:1483-1491; and Mueller et al. (1997) Mol Immunol 34(6):441-452). Forexample (and in accordance with Kabat numbering), the IgG1 and IgG4constant regions contain G₂₄₉G₂₅₀ residues whereas the IgG2 constantregion does not contain residue 249, but does contain G₂₅₀. In a G2/G4hybrid constant region, where the 249-250 region comes from the G2sequence, the constant region can be further modified to introduce aglycine residue at position 249 to produce a G2/G4 fusion havingG₂₄₉/G₂₅₀. Additional methods for eliminating effector function aredescribed below.

It is understood that any of the above-described anti-CD200 antibodiescan be incorporated into the bispecific anti-CD200/anti-CD20 antibodiesdescribed herein.

Anti-CD20 Therapeutic Agents

The disclosure also features therapeutic agents that specifically targetcells (e.g., cancer cells) that express CD20 protein by specificallybinding to CD20 on the surface of the cells. The anti-CD20 therapeuticcan be, e.g., a small molecule compound that binds to CD20, a protein(e.g., a natural or synthetic ligand for CD20) or fragment thereof, anRNA aptamer, an L-RNA aptamer, or a spiegelmer.

In some embodiments, the anti-CD20 therapeutic agents are antibodiesthat bind to CD20 polypeptides (sometimes the antibodies are referred toherein as “anti-CD20 antibodies”). Also featured are antigen-binding(CD20-binding) fragments of the antibodies. In some embodiments, ananti-CD20 antibody described herein binds to an epitope in the humanCD20 protein. For example, the anti-CD20 antibody can bind to an epitopein the human CD20 protein comprising, or consisting of, the followingamino acid sequence:MTTPRNSVNGTFPAEPMKGPIAMQSGPKPLFRRMSSLVGPTQSFFMRESKTLGAVQIMNGLFHIALGGLLMIPAGIYAPICVTVWYPLWGGIMYIISGSLLAATEKNSRKCLVKGKMIMNSLSLFAAISGMILSIMDILNIKISHFLKMESLNFIRAHTPYINIYNCEPANPSEKNSPSTQYCYSIQSLFLGILSVMLIFAFFQELVIAGIVENEWKRTCSRPKSNIVLLSAEEKKEQTIEIKEEVVGLTETSSQPKNEEDIEIIPIQEEEEEETETNFPEPPQDQESSPIENDSSP (SEQ ID NO:40; Genbank Accession No.NP_(—)068769.2). SEQ ID NO:40 depicts the amino acid sequence for afull-length, precursor human CD20 isoform A protein. The amino acidsequence for a full-length human CD20 polypeptide is also described in,e.g., Tedder et al. (1988) Proc Natl Acad Sci USA 85(1):208-212.

An anti-CD20 antibody described herein binds to an epitope within theextracellular portion of a CD20 protein. For example, in someembodiments, the anti-CD20 antibody can bind to CD20 protein at anepitope within or overlapping with: (i) amino acids 72 to 80 of theamino acid sequence depicted in SEQ ID NO:40; or (ii) amino acids 140 to186 of the amino acid sequence depicted in SEQ ID NO:40. See, e.g.,Teeling et al. (2006) J Immunol 177:362-367. That is, an anti-CD20antibody described herein can bind to an epitope of human CD20 within oroverlapping with the amino acid sequence IPAGIYAPI (SEQ ID NO:41) orNIKISHFLKMESLNFIRAHTPYINIYNCEPANPSEKNSPSTQYCYSI (SEQ ID NO:42).

In some embodiments, the anti-CD20 antibody specifically binds to ahuman CD20 protein (e.g., the human CD20 protein having the amino acidsequence depicted in SEQ ID NO:40 or one or more of the extracellularloops of the human CD20 protein). Examples of antibodies thatspecifically bind to a human CD20 protein are described in, e.g.,Teeling et al. (2006) at 363, supra; Levene et al. (2005) JR Soc Med98:146-152; and U.S. Pat. Nos. 7,435,803; 5,595,721; and 7,422,739, thedisclosures of each of which are incorporated herein by reference intheir entirety.

Exemplary therapeutic anti-CD20 antibodies, which are approved forclinical use or are in clinical development, that can be used in themethods described herein include, without limitation, rituximab (BiogenIdec), ⁹⁰Y-ibntumomab tiuxetan (Biogen Idec), ¹³¹I-tositumomab(GlaxoSmithKline), ofatumumab (Genmab), TRU-015 (Trubion), veltuzumab(IMMU-106; Immunomedics), ocrelizumab (Roche), and AME-133v (AppliedMolecular Evolution). See, e.g., Levene et al. (2005), supra; Burge etal. (2008) Clin Ther 30(10):1806-1816; Kausar et al. (2009) Expert OpinBiol Ther 9(7):889-895; Morschhauser et al. (2009) J Clin Oncol27(20):3346-3353; and Milani and Castillo (2009) Curr Opin Mol Ther11(2):200-207.

Methods for determining whether an antibody binds to CD20 and/or theaffinity of an antibody for CD20 are known in the art. In someembodiments, the anti-CD20 antibody can crossblock binding of anotherantibody that binds to an epitope within, or overlapping with, a humanCD20 protein. In some embodiments, the anti-CD20 antibody can crossblockbinding of an antibody that binds to an epitope within, or overlappingwith, a peptide fragment of a human CD20 protein. The peptide fragmentcan be a fragment of a human CD200 protein having the amino acidsequence depicted in, e.g., any one of SEQ ID NO:41 or SEQ ID NO:42.

It is understood that any of the above-described anti-CD200 antibodiescan be incorporated into the bispecific anti-CD200/anti-CD20 antibodiesdescribed herein.

Pharmaceutical Compositions and Formulations

The compositions containing an anti-CD200 antibody, an anti-CD20therapeutic agent such as an anti-CD20 antibody, or both, can beformulated as a pharmaceutical composition, e.g., for administration toa human to treat cancer or an autoimmune disorder. The pharmaceuticalcompositions will generally include a pharmaceutically acceptablecarrier. As used herein, a “pharmaceutically acceptable carrier” refersto, and includes, any and all solvents, dispersion media, coatings,antibacterial and antifungal agents, isotonic and absorption delayingagents, and the like that are physiologically compatible. Thecompositions can include a pharmaceutically acceptable salt, e.g., anacid addition salt or a base addition salt. See, e.g., Berge et al.(1977) J Pharm Sci 66:1-19.

The compositions can be formulated according to standard methods.Pharmaceutical formulation is a well-established art, and is furtherdescribed in, e.g., Gennaro (2000) “Remington: The Science and Practiceof Pharmacy,” 20^(th) Edition, Lippincott, Williams & Wilkins (ISBN:0683306472); Ansel et al. (1999) “Pharmaceutical Dosage Forms and DrugDelivery Systems,” 7^(th) Edition, Lippincott Williams & WilkinsPublishers (ISBN: 0683305727); and Kibbe (2000) “Handbook ofPharmaceutical Excipients American Pharmaceutical Association,” 3^(rd)Edition (ISBN: 091733096X). In some embodiments, a composition can beformulated, for example, as a buffered solution at a suitableconcentration and suitable for storage at 2-8° C. In some embodiments, acomposition can be formulated for storage at a temperature below 0° C.(e.g., −20° C. or −80° C.).

The pharmaceutical compositions can be in a variety of forms. Theseforms include, e.g., liquid, semi-solid and solid dosage forms, such asliquid solutions (e.g., injectable and infusible solutions), dispersionsor suspensions, tablets, pills, powders, liposomes and suppositories.The preferred form depends, in part, on the intended mode ofadministration and therapeutic application. For example, compositionscontaining an anti-CD200 antibody or an anti-CD20 antibody, intended forsystemic or local delivery can be in the form of injectable or infusiblesolutions. Accordingly, the compositions can be formulated foradministration by a parenteral mode (e.g., intravenous, subcutaneous,intraperitoneal, or intramuscular injection). “Parenteraladministration,” “administered parenterally,” and other grammaticallyequivalent phrases, as used herein, refer to modes of administrationother than enteral and topical administration, usually by injection, andinclude, without limitation, intravenous, intranasal, intraocular,pulmonary, intramuscular, intraarterial, intrathecal, intracapsular,intraorbital, intracardiac, intradermal, intrapulmonary,intraperitoneal, transtracheal, subcutaneous, subcuticular,intraarticular, subcapsular, subarachnoid, intraspinal, epidural,intracerebral, intracranial, intracarotid and intrasternal injection andinfusion (see below).

The compositions can be formulated as a solution, microemulsion,dispersion, liposome, or other ordered structure suitable for stablestorage at high concentration. Sterile injectable solutions can beprepared by incorporating an antibody described herein in the requiredamount in an appropriate solvent with one or a combination ofingredients enumerated above, as required, followed by filteredsterilization. Generally, dispersions are prepared by incorporating ananti-CD200 antibody (and/or an anti-CD20 therapeutic agent such as ananti-CD20 antibody) described herein into a sterile vehicle thatcontains a basic dispersion medium and the required other ingredientsfrom those enumerated above. In the case of sterile powders for thepreparation of sterile injectable solutions, methods for preparationinclude vacuum drying and freeze-drying that yield a powder of theantibody described herein plus any additional desired ingredient from apreviously sterile-filtered solution thereof. The proper fluidity of asolution can be maintained, for example, by the use of a coating such aslecithin, by the maintenance of the required particle size in the caseof dispersion and by the use of surfactants. Prolonged absorption ofinjectable compositions can be brought about by including in thecomposition a reagent that delays absorption, for example, monostearatesalts and gelatin.

In certain embodiments, the anti-CD200 antibody (and/or the anti-CD20therapeutic agent such as an anti-CD20 antibody) can be prepared with acarrier that will protect the compound against rapid release, such as acontrolled release formulation, including implants and microencapsulateddelivery systems. Biodegradable, biocompatible polymers can be used,such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid,collagen, polyorthoesters, and polylactic acid. Many methods for thepreparation of such formulations are known in the art. (See, e.g., J. R.Robinson (1978) “Sustained and Controlled Release Drug DeliverySystems,” Marcel Dekker, Inc., New York.)

In some embodiments, an antibody described herein can be formulated in acomposition suitable for intrapulmonary administration (e.g., foradministration via nebulizer) to a mammal such as a human. Methods forpreparing such compositions are well known in the art and described in,e.g., U.S. Patent Application Publication No. 20080202513; U.S. Pat.Nos. 7,112,341 and 6,019,968; and PCT Publication Nos. WO 00/061178 andWO 06/122257, the disclosures of each of which are incorporated hereinby reference in their entirety. Dry powder inhaler formulations andsuitable systems for administration of the formulations are describedin, e.g., U.S. Patent Application Publication No. 20070235029, PCTPublication No. WO 00/69887; and U.S. Pat. No. 5,997,848.

In some embodiments, an anti-CD200 antibody (and/or an anti-CD20therapeutic agent such as an anti-CD20 antibody) described herein can bemodified, e.g., with a moiety that improves its stabilization and/orretention in circulation, e.g., in blood, serum, or other tissues. Thestabilization moiety can improve the stability, or retention of, theantibody by at least 1.5 (e.g., at least 2, 5, 10, 15, 20, 25, 30, 40,or 50 or more) fold.

In some embodiments, an anti-CD200 antibody described herein can beformulated with one or more additional active agents useful for treatingcancer or ameliorating a symptom thereof. For example, an anti-CD200antibody can be formulated with an anti-CD20 therapeutic agent (e.g., ananti-CD20 antibody such as any of the anti-CD20 antibodies describedherein), a genotoxic agent or a chemotherapeutic agent, or one or morekinase inhibitors. The genotoxic or chemotherapeutic agent can be, butis not limited to: carboplatin, procarbazine, mechlorethamine,cyclophosphamide, camptothecin, ifosfamide, melphalan, chlorambucil,bisulfan, nitrosurea, dactinomycin, daunorubicin, doxorubicin,bleomycin, plicomycin, mitomycin, etoposide, podophyllotoxin, taxol,satraplatinum, 5-fluorouracil, vincristin, vinblastin, methotrexate,ara-C, taxotere, gemcitabine, cisplatin (CDDP), adriamycin (ADR), or ananalog of any of the aforementioned. Kinase inhibitors include, e.g.,one or more of: trastuzumab, gefitinib, erlotinib, imatinib mesylate, orsunitinib malate. Additional agents are known in the art and describedherein.

When the anti-CD200 antibody is to be used in combination with a secondactive agent, or when two or more different anti-CD200 antibodies are tobe used, the agents can be formulated separately or together. Forexample, the respective pharmaceutical compositions can be mixed, e.g.,just prior to administration, and administered together or can beadministered separately, e.g., at the same or different times (seebelow).

As described above, a composition can be formulated such that itincludes a therapeutically effective amount of an anti-CD200 antibody orthe composition can be formulated to include a sub-therapeutic amount ofthe antibody and a sub-therapeutic amount of one or more additionalactive agents such that the components in total are therapeuticallyeffective for treating a cancer or an autoimmune disorder. In someembodiments, a composition can be formulated to include two or moreanti-CD200 antibodies, each at sub-therapeutic doses, such that theantibodies in combination are at a concentration that is therapeuticallyeffective for treating a cancer or an autoimmune disorder in a human.Methods for determining a therapeutically effective dose of ananti-CD200 antibody are known in the art and described herein.

Methods for Producing an Anti-CD200 or an Anti-CD20 Antibody

Suitable methods for producing an antibody (e.g., an anti-CD200 antibodyor an anti-CD20 antibody) or antigen-binding fragments thereof, inaccordance with the disclosure are known in the art (see, e.g., U.S.Pat. Nos. 7,427,665; 7,435,412; and 7,408,041, the disclosures of eachof which are incorporated herein by reference in their entirety) anddescribed herein. For example, monoclonal anti-CD200 antibodies may begenerated using human CD200-expressing cells, a human CD200 polypeptide,or an antigenic fragment of a human CD200 polypeptide as an immunogen,thus raising an immune response in animals from which antibody-producingcells and in turn monoclonal antibodies may be isolated. Similarly, amonoclonal anti-CD20 antibody can be generated using humanCD20-expressing cells, a human CD20 polypeptide, or an antigenicfragment of the human CD20 polypeptide as an immunogen in an animal. Thesequence of such antibodies may be determined and the antibodies orvariants thereof produced by recombinant techniques. Recombinanttechniques may be used to produce chimeric, CDR-grafted, humanized andfully human antibodies based on the sequence of the monoclonalantibodies as well as polypeptides capable of binding to the antigen ofinterest (e.g., CD200 or CD20).

Moreover, anti-CD200 antibodies derived from recombinant libraries(“phage antibodies”) may be selected using CD200-expressing cells, orpolypeptides derived therefrom, as bait to isolate the antibodies orpolypeptides on the basis of target specificity. The production andisolation of non-human and chimeric anti-CD200 antibodies are wellwithin the purview of the skilled artisan. It is understood thatanti-CD20 antibodies can be selected using merely routine adaptations ofthe methods described above.

Recombinant DNA technology can be used to modify one or morecharacteristics of the antibodies produced in non-human cells. Thus,chimeric antibodies can be constructed in order to decrease theimmunogenicity thereof in diagnostic or therapeutic applications.Moreover, immunogenicity can be minimized by humanizing the antibodiesby CDR grafting and, optionally, framework modification. See, U.S. Pat.Nos. 5,225,539 and 7,393,648, the contents of each of which areincorporated herein by reference.

Antibodies can be obtained from animal serum or, in the case ofmonoclonal antibodies or fragments thereof, produced in cell culture.Recombinant DNA technology can be used to produce the antibodiesaccording to established procedure, including procedures in bacterial orpreferably mammalian cell culture. The selected cell culture systempreferably secretes the antibody product.

In another embodiment, a process for the production of an antibodydisclosed herein includes culturing a host, e.g. E. coli or a mammaliancell, which has been transformed with a hybrid vector. The vectorincludes one or more expression cassettes containing a promoter operablylinked to a first DNA sequence encoding a signal peptide linked in theproper reading frame to a second DNA sequence encoding the antibodyprotein. The antibody protein is then collected and isolated.Optionally, the expression cassette may include a promoter operablylinked to a polycistronic (e.g., bicistronic) DNA sequence encodingantibody proteins each individually operably linked to a signal peptidein the proper reading frame.

Multiplication of hybridoma cells or mammalian host cells in vitro iscarried out in suitable culture media, which include the customarystandard culture media (such as, for example Dulbecco's Modified EagleMedium (DMEM) or RPMI 1640 medium), optionally replenished by amammalian serum (e.g. fetal calf serum), or trace elements and growthsustaining supplements (e.g. feeder cells such as normal mouseperitoneal exudate cells, spleen cells, bone marrow macrophages,2-aminoethanol, insulin, transferrin, low density lipoprotein, oleicacid, or the like). Multiplication of host cells which are bacterialcells or yeast cells is likewise carried out in suitable culture mediaknown in the art. For example, for bacteria suitable culture mediainclude medium LE, NZCYM, NZYM, NZM, Terrific Broth, SOB, SOC, 2×YT, orM9 Minimal Medium. For yeast, suitable culture media include medium YPD,YEPD, Minimal Medium, or Complete Minimal Dropout Medium.

In vitro production provides relatively pure antibody preparations andallows scale-up production to give large amounts of the desiredantibodies. Techniques for bacterial cell, yeast, plant, or mammaliancell cultivation are known in the art and include homogeneous suspensionculture (e.g. in an airlift reactor or in a continuous stirrer reactor),and immobilized or entrapped cell culture (e.g. in hollow fibers,microcapsules, on agarose microbeads or ceramic cartridges).

Large quantities of the desired antibodies can also be obtained bymultiplying mammalian cells in vivo. For this purpose, hybridoma cellsproducing the desired antibodies are injected into histocompatiblemammals to cause growth of antibody-producing tumors. Optionally, theanimals are primed with a hydrocarbon, especially mineral oils such aspristane (tetramethyl-pentadecane), prior to the injection. After one tothree weeks, the antibodies are isolated from the body fluids of thosemammals. For example, hybridoma cells obtained by fusion of suitablemyeloma cells with antibody-producing spleen cells from Balb/c mice, ortransfected cells derived from hybridoma cell line Sp2/0 that producethe desired antibodies are injected intraperitoneally into Balb/c miceoptionally pre-treated with pristane. After one to two weeks, asciticfluid is taken from the animals.

The foregoing, and other, techniques are discussed in, for example,Kohler and Milstein, (1975) Nature 256:495-497; U.S. Pat. No. 4,376,110;Harlow and Lane, Antibodies: a Laboratory Manual, (1988) Cold SpringHarbor, the disclosures of which are all incorporated herein byreference. Techniques for the preparation of recombinant antibodymolecules are described in the above references and also in, e.g.:WO97/08320; U.S. Pat. No. 5,427,908; U.S. Pat. No. 5,508,717; Smith(1985) Science 225:1315-1317; Parmley and Smith (1988) Gene 73:305-318;De La Cruz et al. (1988) Journal of Biological Chemistry 263:4318-4322;U.S. Pat. No. 5,403,484; U.S. Pat. No. 5,223,409; WO88/06630;WO92/15679; U.S. Pat. No. 5,780,279; U.S. Pat. No. 5,571,698; U.S. Pat.No. 6,040,136; Davis et al. (1999) Cancer Metastasis Rev. 18(4):421-5;and Taylor et al. (1992) Nucleic Acids Research 20: 6287-6295; Tomizukaet al. (2000) Proc. Natl. Acad. Sci. USA 97(2): 722-727, the contents ofeach of which are incorporated herein by reference in their entirety.

The cell culture supernatants are screened for the desired antibodies,preferentially by immunofluorescent staining of CD200-expressing cells,by immunoblotting, by an enzyme immunoassay, e.g. a sandwich assay or adot-assay, or a radioimmunoassay.

For isolation of the antibodies, the immunoglobulins in the culturesupernatants or in the ascitic fluid may be concentrated, e.g., byprecipitation with ammonium sulfate, dialysis against hygroscopicmaterial such as polyethylene glycol, filtration through selectivemembranes, or the like. If necessary and/or desired, the antibodies arepurified by the customary chromatography methods, for example gelfiltration, ion-exchange chromatography, chromatography overDEAE-cellulose and/or (immuno-) affinity chromatography, e.g., affinitychromatography with one or more surface polypeptides derived from aCD200-expressing cell line or synthetic CD200 fragment peptides, or withProtein-A or -G.

Another embodiment provides a process for the preparation of a bacterialcell line secreting antibodies directed against a human CD200 protein ora human CD20 (depending on the antibody being generated) in a suitablemammal. For example a rabbit is immunized with pooled samples fromCD200-expressing tissue or cells or CD200 polypeptide or fragmentsthereof. A phage display library produced from the immunized rabbit isconstructed and panned for the desired antibodies in accordance withmethods well known in the art (such as, e.g., the methods disclosed inthe various references incorporated herein by reference).

Hybridoma cells secreting the monoclonal antibodies are also disclosed.The preferred hybridoma cells are genetically stable, secrete monoclonalantibodies described herein of the desired specificity, and can beexpanded from deep-frozen cultures by thawing and propagation in vitroor as ascites in vivo.

In another embodiment, a process is provided for the preparation of ahybridoma cell line secreting monoclonal antibodies against a humanCD200 protein. In that process, a suitable mammal, for example a Balb/cmouse, is immunized with one or more polypeptides or antigenic fragmentsof CD200 or with one or more polypeptides or antigenic fragments derivedfrom a CD200-expressing cell, the CD200-expressing cell itself, or anantigenic carrier containing a purified polypeptide as described.Antibody-producing cells of the immunized mammal are grown briefly inculture or fused with cells of a suitable myeloma cell line. The hybridcells obtained in the fusion are cloned, and cell clones secreting thedesired antibodies are selected. For example, spleen cells of Balb/cmice immunized with a protein fragment of human CD200 are fused withcells of the myeloma cell line PAI or the myeloma cell line Sp2/0-Ag 14.The obtained hybrid cells are then screened for secretion of the desiredantibodies and positive hybridoma cells are cloned.

Methods for preparing a hybridoma cell line include immunizing Balb/cmice by injecting subcutaneously and/or intraperitoneally a peptidefragment of human CD200 several times, e.g., four to six times, overseveral months, e.g., between two and four months. Spleen cells from theimmunized mice are taken two to four days after the last injection andfused with cells of the myeloma cell line PAI in the presence of afusion promoter, preferably polyethylene glycol. Preferably, the myelomacells are fused with a three- to twenty-fold excess of spleen cells fromthe immunized mice in a solution containing about 30% to about 50%polyethylene glycol of a molecular weight around 4000. After the fusion,the cells are expanded in suitable culture media as described supra,supplemented with a selection medium, for example HAT medium, at regularintervals in order to prevent normal myeloma cells from overgrowing thedesired hybridoma cells.

The antibodies and fragments thereof can be “chimeric.” Chimericantibodies and antigen-binding fragments thereof comprise portions fromtwo or more different species (e.g., mouse and human). Chimericantibodies can be produced with mouse variable regions of desiredspecificity spliced into human constant domain gene segments (forexample, U.S. Pat. No. 4,816,567). In this manner, non-human antibodiescan be modified to make them more suitable for human clinicalapplication (e.g., methods for treating or preventing a cancer in ahuman subject).

The monoclonal antibodies of the present disclosure include “humanized”forms of the non-human (e.g., mouse) antibodies. Humanized orCDR-grafted mAbs are particularly useful as therapeutic agents forhumans because they are not cleared from the circulation as rapidly asmouse antibodies and do not typically provoke an adverse immunereaction. Generally, a humanized antibody has one or more amino acidresidues introduced into it from a non-human source. These non-humanamino acid residues are often referred to as “import” residues, whichare typically taken from an “import” variable domain. Methods ofpreparing humanized antibodies are generally well known in the art. Forexample, humanization can be essentially performed following the methodof Winter and co-workers (see, e.g., Jones et al. (1986) Nature321:522-525; Riechmann et al. (1988) Nature 332:323-327; and Verhoeyenet al. (1988) Science 239:1534-1536), by substituting rodent CDRs or CDRsequences for the corresponding sequences of a human antibody. Also see,e.g., Staelens et al. (2006) Mol Immunol 43:1243-1257. In someembodiments, humanized forms of non-human (e.g., mouse) antibodies arehuman antibodies (recipient antibody) in which hypervariable (CDR)region residues of the recipient antibody are replaced by hypervariableregion residues from a non-human species (donor antibody) such as amouse, rat, rabbit, or non-human primate having the desired specificity,affinity, and binding capacity. In some instances, framework regionresidues of the human immunoglobulin are also replaced by correspondingnon-human residues (so called “back mutations”). In addition, phagedisplay libraries can be used to vary amino acids at chosen positionswithin the antibody sequence. The properties of a humanized antibody arealso affected by the choice of the human framework. Furthermore,humanized and chimerized antibodies can be modified to comprise residuesthat are not found in the recipient antibody or in the donor antibody inorder to further improve antibody properties, such as, for example,affinity or effector function.

Fully human antibodies are also provided in the disclosure. The term“human antibody” includes antibodies having variable and constantregions (if present) derived from human germline immunoglobulinsequences. Human antibodies can include amino acid residues not encodedby human germline immunoglobulin sequences (e.g., mutations introducedby random or site-specific mutagenesis in vitro or by somatic mutationin vivo). However, the term “human antibody” does not include antibodiesin which CDR sequences derived from the germline of another mammalianspecies, such as a mouse, have been grafted onto human frameworksequences (i.e., humanized antibodies). Fully human or human antibodiesmay be derived from transgenic mice carrying human antibody genes(carrying the variable (V), diversity (D), joining (J), and constant (C)exons) or from human cells. For example, it is now possible to producetransgenic animals (e.g., mice) that are capable, upon immunization, ofproducing a full repertoire of human antibodies in the absence ofendogenous immunoglobulin production. See, e.g., Jakobovits et al.(1993) Proc Natl Acad Sci USA 90:2551; Jakobovits et al. (1993) Nature362:255-258; Bruggemann et al. (1993) Year in Immunol 7:33; and Duchosalet al. (1992) Nature 355:258. Transgenic mouse strains can be engineeredto contain gene sequences from unrearranged human immunoglobulin genes.The human sequences may code for both the heavy and light chains ofhuman antibodies and would function correctly in the mice, undergoingrearrangement to provide a wide antibody repertoire similar to that inhumans. The transgenic mice can be immunized with the target protein(e.g., a human CD200 protein, fragments thereof, or cells expressingCD200 protein; or a human CD20 protein, fragments thereof, or cellsexpressing CD20 protein) to create a diverse array of specificantibodies and their encoding RNA. Nucleic acids encoding the antibodychain components of such antibodies may then be cloned from the animalinto a display vector. Typically, separate populations of nucleic acidsencoding heavy and light chain sequences are cloned, and the separatepopulations then recombined on insertion into the vector, such that anygiven copy of the vector receives a random combination of a heavy and alight chain. The vector is designed to express antibody chains so thatthey can be assembled and displayed on the outer surface of a displaypackage containing the vector. For example, antibody chains can beexpressed as fusion proteins with a phage coat protein from the outersurface of the phage. Thereafter, display packages can be screened fordisplay of antibodies binding to a target.

In addition, human antibodies can be derived from phage-displaylibraries (Hoogenboom et al. (1991) J Mol Biol 227:381; Marks et al.(1991) J Mol Biol 222:581-597; and Vaughan et al. (1996) Nature Biotech14:309 (1996)). Synthetic phage libraries can be created which userandomized combinations of synthetic human antibody V-regions. Byselection on antigen fully human antibodies can be made in which theV-regions are very human-like in nature. See, e.g., U.S. Pat. Nos.6,794,132, 6,680,209, 4,634,666, and Ostberg et al. (1983) Hybridoma2:361-367, the contents of each of which are incorporated herein byreference in their entirety.

For the generation of human antibodies, also see Mendez et al. (1998)Nature Genetics 15:146-156, Green and Jakobovits (1998) J Exp Med188:483-495, the disclosures of which are hereby incorporated byreference in their entirety. Human antibodies are further discussed anddelineated in U.S. Pat. Nos. 5,939,598; 6,673,986; 6,114,598; 6,075,181;6,162,963; 6,150,584; 6,713,610; and 6,657,103 as well as U.S. PatentPublication Nos. 20030229905 A1, 20040010810 A1, US 20040093622 A1,20060040363 A1, 20050054055 A1, 20050076395 A1, 20050287630 A1. See alsoInternational Publication Nos. WO 94/02602, WO 96/34096, and WO98/24893, and European Patent No. EP 0 463 151 B1. The disclosures ofeach of the above-cited patents, applications, and references are herebyincorporated by reference in their entirety.

In an alternative approach, others, including GenPharm International,Inc., have utilized a “minilocus” approach. In the minilocus approach,an exogenous Ig locus is mimicked through the inclusion of pieces(individual genes) from the Ig locus. Thus, one or more V_(H) genes, oneor more D_(H) genes, one or more J_(H) genes, a mu constant region, anda second constant region (preferably a gamma constant region) are formedinto a construct for insertion into an animal. This approach isdescribed in, e.g., U.S. Pat. Nos. 5,545,807; 5,545,806; 5,625,825;5,625,126; 5,633,425; 5,661,016; 5,770,429; 5,789,650; and 5,814,318;5,591,669; 5,612,205; 5,721,367; 5,789,215; 5,643,763; 5,569,825;5,877,397; 6,300,129; 5,874,299; 6,255,458; and 7,041,871, thedisclosures of which are hereby incorporated by reference. See alsoEuropean Patent No. 0 546 073 B1, International Patent Publication Nos.WO 92/03918, WO 92/22645, WO 92/22647, WO 92/22670, WO 93/12227, WO94/00569, WO 94/25585, WO 96/14436, WO 97/13852, and WO 98/24884, thedisclosures of each of which are hereby incorporated by reference intheir entirety. See further Taylor et al. (1992) Nucleic Acids Res 20:6287; Chen et al. (1993) Int Immunol 5: 647; Tuaillon et al. (1993) ProcNatl Acad Sci USA 90: 3720-4; Choi et al. (1993) Nature Genetics 4: 117;Lonberg et al. (1994) Nature 368: 856-859; Taylor et al. (1994)International Immunology 6: 579-591; Tuaillon et al. (1995) J. Immunol.154: 6453-65; Fishwild et al. (1996) Nature Biotechnology 14: 845; andTuaillon et al. (2000) Eur J Immunol 10: 2998-3005, the disclosures ofeach of which are hereby incorporated by reference in their entirety.

In certain embodiments, de-immunized anti-CD200 antibodies orantigen-binding fragments thereof are provided. De-immunized antibodiesor antigen-binding fragments thereof are those modified so as to renderthe antibody or antigen-binding fragment thereof non-immunogenic, orless immunogenic, to a given species. De-immunization can be achieved bymodifying the antibody or antigen-binding fragment thereof utilizing anyof a variety of techniques known to those skilled in the art (see, e.g.,PCT Publication Nos. WO 04/108158 and WO 00/34317). For example, anantibody or antigen-binding fragment thereof may be de-immunized byidentifying potential T cell epitopes and/or B cell epitopes within theamino acid sequence of the antibody or antigen-binding fragment thereofand removing one or more of the potential T cell epitopes and/or B cellepitopes from the antibody or antigen-binding fragment thereof, forexample, using recombinant techniques. The modified antibody orantigen-binding fragment thereof may then optionally be produced andtested to identify antibodies or antigen-binding fragments thereof thathave retained one or more desired biological activities, such as, forexample, binding affinity, but have reduced immunogenicity. Methods foridentifying potential T cell epitopes and/or B cell epitopes may becarried out using techniques known in the art, such as, for example,computational methods (see e.g., PCT Publication No. WO 02/069232), invitro or in silico techniques, and biological assays or physical methods(such as, for example, determination of the binding of peptides to MHCmolecules, determination of the binding of peptide:MHC complexes to theT cell receptors from the species to receive the antibody orantigen-binding fragment thereof, testing of the protein or peptideparts thereof using transgenic animals with the MHC molecules of thespecies to receive the antibody or antigen-binding fragment thereof, ortesting with transgenic animals reconstituted with immune system cellsfrom the species to receive the antibody or antigen-binding fragmentthereof, etc.). In various embodiments, the de-immunized antibodies(e.g., deimmunized anti-CD200 antibodies or deimmunized anti-CD20antibodies) described herein include de-immunized antigen-bindingfragments, Fab, Fv, scFv, Fab′ and F(ab′)₂, monoclonal antibodies,murine antibodies, engineered antibodies (such as, for example,chimeric, single chain, CDR-grafted, humanized, fully human antibodies,and artificially selected antibodies), synthetic antibodies andsemi-synthetic antibodies.

In some embodiments, a recombinant DNA comprising an insert coding for aheavy chain variable domain and/or for a light chain variable domain ofan anti-CD200 antibody or a CD200 protein-expressing cell line isproduced. The term DNA includes coding single stranded DNAs, doublestranded DNAs consisting of said coding DNAs and of complementary DNAsthereto, or these complementary (single stranded) DNAs themselves.

Furthermore, a DNA encoding a heavy chain variable domain and/or a lightchain variable domain of anti-CD200 antibodies, or the CD200-expressingcell line, can be enzymatically or chemically synthesized to contain theauthentic DNA sequence coding for a heavy chain variable domain and/orfor the light chain variable domain, or a mutant thereof. A mutant ofthe authentic DNA is a DNA encoding a heavy chain variable domain and/ora light chain variable domain of the above-mentioned antibodies in whichone or more amino acids are deleted, inserted, or exchanged with one ormore other amino acids. Preferably said modification(s) are outside theCDRs of the heavy chain variable domain and/or of the light chainvariable domain of the antibody in humanization and expressionoptimization applications. The term mutant DNA also embraces silentmutants wherein one or more nucleotides are replaced by othernucleotides with the new codons coding for the same amino acid(s). Theterm mutant sequence also includes a degenerate sequence. Degeneratesequences are degenerate within the meaning of the genetic code in thatan unlimited number of nucleotides are replaced by other nucleotideswithout resulting in a change of the amino acid sequence originallyencoded. Such degenerate sequences may be useful due to their differentrestriction sites and/or frequency of particular codons which arepreferred by the specific host, particularly E. coli, to obtain anoptimal expression of the heavy chain murine variable domain and/or alight chain murine variable domain.

The term mutant is intended to include a DNA mutant obtained by in vitromutagenesis of the authentic DNA according to methods known in the art.

For the assembly of complete tetrameric immunoglobulin molecules and theexpression of chimeric antibodies, the recombinant DNA inserts codingfor heavy and light chain variable domains are fused with thecorresponding DNAs coding for heavy and light chain constant domains,then transferred into appropriate host cells, for example afterincorporation into hybrid vectors.

Recombinant DNAs including an insert coding for a heavy chain murinevariable domain of an anti-CD200 antibody or a CD200-expressing cellline fused to a human constant domain IgG, for example γ1, γ2, γ3 or γ4,in particular embodiments γ1 or γ4, may be used. Recombinant DNAsincluding an insert coding for a light chain murine variable domain ofan antibody fused to a human constant domain κ or λ, preferably κ, arealso provided.

Another embodiment pertains to recombinant DNAs coding for a recombinantpolypeptide wherein the heavy chain variable domain and the light chainvariable domain are linked by way of a spacer group, optionallycomprising a signal sequence facilitating the processing of the antibodyin the host cell and/or a DNA sequence encoding a peptide facilitatingthe purification of the antibody and/or a cleavage site and/or a peptidespacer and/or an agent. The DNA coding for an agent is intended to be aDNA coding for the agent useful in diagnostic or therapeuticapplications. Thus, agent molecules which are toxins or enzymes,especially enzymes capable of catalyzing the activation of prodrugs, areparticularly indicated. The DNA encoding such an agent has the sequenceof a naturally occurring enzyme or toxin encoding DNA, or a mutantthereof, and can be prepared by methods well known in the art.

Accordingly, the monoclonal antibodies or antigen-binding fragments ofthe disclosure can be naked antibodies or antigen-binding fragments thatare not conjugated to other agents, for example, a therapeutic agent ordetectable label.

Alternatively, the monoclonal antibody or antigen-binding fragment canbe conjugated to an agent such as, for example, a cytotoxic agent, asmall molecule, a hormone, an enzyme, a growth factor, a cytokine, aribozyme, a peptidomimetic, a chemical, a prodrug, a nucleic acidmolecule including coding sequences (such as antisense, RNAi,gene-targeting constructs, etc.), or a detectable label (e.g., an NMR orX-ray contrasting agent, fluorescent molecule, etc.). In certainembodiments, an anti-CD200 antibody or antigen-binding fragment (e.g.,Fab, Fv, single-chain scFv, Fab′, and F(ab′)₂) is linked to a moleculethat increases the half-life of the antibody or antigen-binding fragment(see above).

Several possible vector systems are available for the expression ofcloned heavy chain and light chain genes in mammalian cells. One classof vectors relies upon the integration of the desired gene sequencesinto the host cell genome. Cells which have stably integrated DNA can beselected by simultaneously introducing drug resistance genes such as E.coli gpt (Mulligan and Berg (1981) Proc Natl Acad Sci USA, 78:2072) orTn5 neo (Southern and Berg (1982) Mol Appl Genet. 1:327). The selectablemarker gene can be either linked to the DNA gene sequences to beexpressed, or introduced into the same cell by co-transfection (Wigleret al. (1979) Cell 16:77). A second class of vectors utilizes DNAelements which confer autonomously replicating capabilities to anextrachromosomal plasmid. These vectors can be derived from animalviruses, such as bovine papillomavirus (Sarver et al. (1982) Proc NatlAcad Sci USA, 79:7147), polyoma virus (Deans et al. (1984) Proc NatlAcad Sci USA 81:1292), or SV40 virus (Lusky and Botchan (1981) Nature293:79).

Since an immunoglobulin cDNA is comprised only of sequences representingthe mature mRNA encoding an antibody protein, additional gene expressionelements regulating transcription of the gene and processing of the RNAare required for the synthesis of immunoglobulin mRNA. These elementsmay include splice signals, transcription promoters, including induciblepromoters, enhancers, and termination signals. cDNA expression vectorsincorporating such elements include those described by Okayama and Berg(1983) Mol Cell Biol 3:280; Cepko et al. (1984) Cell 37:1053; andKaufman (1985) Proc Natl Acad Sci USA 82:689.

As is evident from the disclosure, the anti-CD200 antibodies and/oranti-CD20 antibodies can be used in therapies (e.g., therapies fortreating a cancer), including combination therapies, as well as in themonitoring of disease progression.

In the therapeutic embodiments of the present disclosure, bispecificantibodies are contemplated. Bispecific antibodies are monoclonal,preferably human or humanized, antibodies that have bindingspecificities for at least two different antigens. In the present case,one of the binding specificities is for the CD200 antigen on a cell(such as, e.g., an immune cell), the other one is for any other antigen,and preferably for a cell-surface protein or receptor or receptorsubunit. In some embodiments, the bispecific antibody is one that bindsto human CD200 and human CD20.

Methods for making bispecific antibodies are within the purview of thoseskilled in the art. Traditionally, the recombinant production ofbispecific antibodies is based on the co-expression of twoimmunoglobulin heavy-chain/light-chain pairs, where the two heavy chainshave different specificities (Milstein and Cuello (1983) Nature305:537-539). Antibody variable domains with the desired bindingspecificities (antibody-antigen combining sites) can be fused toimmunoglobulin constant domain sequences. The fusion preferably is withan immunoglobulin heavy-chain constant domain, including at least partof the hinge, C_(H)2, and C_(H)3 regions. DNAs encoding theimmunoglobulin heavy-chain fusions and, if desired, the immunoglobulinlight chain, are inserted into separate expression vectors, and areco-transfected into a suitable host organism. For further details ofillustrative currently known methods for generating bispecificantibodies see, e.g., Suresh et al. (1986) Methods in Enzymology121:210; PCT Publication No. WO 96/27011; Brennan et al. (1985) Science229:81; Shalaby et al. J Exp Med (1992) 175:217-225; Kostelny et al.(1992) J Immunol 148(5):1547-1553; Hollinger et al. (1993) Proc. Natl.Acad. Sci. USA 90:6444-6448; Gruber et al. (1994) J Immunol 152:5368;and Tutt et al. (1991) J Immunol 147:60. Bispecific antibodies alsoinclude cross-linked or heteroconjugate antibodies. Heteroconjugateantibodies may be made using any convenient cross-linking methods.Suitable cross-linking agents are well known in the art, and aredisclosed in U.S. Pat. No. 4,676,980, along with a number ofcross-linking techniques.

Various techniques for making and isolating bispecific antibodyfragments directly from recombinant cell culture have also beendescribed. For example, bispecific antibodies have been produced usingleucine zippers. See, e.g., Kostelny et al. (1992) J Immunol148(5):1547-1553. The leucine zipper peptides from the Fos and Junproteins may be linked to the Fab′ portions of two different antibodiesby gene fusion. The antibody homodimers may be reduced at the hingeregion to form monomers and then re-oxidized to form the antibodyheterodimers. This method can also be utilized for the production ofantibody homodimers. The “diabody” technology described by Hollinger etal. (1993) Proc Natl Acad Sci USA 90:6444-6448 has provided analternative mechanism for making bispecific antibody fragments. Thefragments comprise a heavy-chain variable domain (VH) connected to alight-chain variable domain (VL) by a linker which is too short to allowpairing between the two domains on the same chain. Accordingly, the VHand VL domains of one fragment are forced to pair with the complementaryVL and VH domains of another fragment, thereby forming twoantigen-binding sites. Another strategy for making bispecific antibodyfragments by the use of single-chain Fv (scFv) dimers has also beenreported. See, e.g., Gruber et al. (1994) J Immunol 152:5368.Alternatively, the antibodies can be “linear antibodies” as describedin, e.g., Zapata et al. (1995) Protein Eng 8(10):1057-1062. Briefly,these antibodies comprise a pair of tandem Fd segments(V_(H)-C_(H)1-V_(H)-C_(H)1) which form a pair of antigen bindingregions. Linear antibodies can be bispecific or monospecific.

The disclosure also embraces variant forms of bispecific antibodies suchas the tetravalent dual variable domain immunoglobulin (DVD-Ig)molecules described in Wu et al. (2007) Nat Biotechnol 25(11):1290-1297.The DVD-Ig molecules are designed such that two different light chainvariable domains (VL) from two different parent antibodies are linked intandem directly or via a short linker by recombinant DNA techniques,followed by the light chain constant domain. Methods for generatingDVD-Ig molecules from two parent antibodies are further described in,e.g., PCT Publication Nos. WO 08/024,188 and WO 07/024,715, thedisclosures of each of which are incorporated herein by reference intheir entirety.

In some embodiments, anti-CD200 antibodies and/or anti-CD20 antibodiescan be modified, e.g., with a moiety that improves the stabilizationand/or retention of the antibodies themselves in circulation, e.g., inblood, serum, or other tissues. For example, an anti-CD200 antibodydescribed herein can be PEGylated as described in, e.g., Lee et al.(1999) Bioconjug Chem 10(6): 973-8; Kinstler et al. (2002) Advanced DrugDeliveries Reviews 54:477-485; and Roberts et al. (2002) Advanced DrugDelivery Reviews 54:459-476. The stabilization moiety can improve thestability, or retention of, the antibody in a subject's body (e.g.,blood or tissue) by at least 1.5 (e.g., at least 2, 5, 10, 15, 20, 25,30, 40, or 50 or more) fold.

Modification of the Antibodies or Antigen-Binding Fragments Thereof

The anti-CD200 antibodies or anti-CD20 antibodies, or antigen-bindingfragments thereof, can be modified following their expression andpurification. The modifications can be covalent or non-covalentmodifications. Such modifications can be introduced into the antibodiesor fragments by, e.g., reacting targeted amino acid residues of thepolypeptide with an organic derivatizing agent that is capable ofreacting with selected side chains or terminal residues. Suitable sitesfor modification can be chosen using any of a variety of criteriaincluding, e.g., structural analysis or amino acid sequence analysis ofthe antibodies or fragments.

In some embodiments, the antibodies or antigen-binding fragments thereofcan be conjugated to a heterologous moiety. The heterologous moiety canbe, e.g., a heterologous polypeptide, a therapeutic agent (e.g., a toxinor a drug), or a detectable label such as, but not limited to, aradioactive label, an enzymatic label, a fluorescent label, or aluminescent label. Suitable heterologous polypeptides include, e.g., anantigenic tag (e.g., FLAG, polyhistidine, hemagglutinin (HA),glutathione-S-transferase (GST), or maltose-binding protein (MBP)) foruse in purifying the antibodies or fragments. Heterologous polypeptidesalso include polypeptides that are useful as diagnostic or detectablemarkers, for example, luciferase, green fluorescent protein (GFP), orchloramphenicol acetyl transferase (CAT). Suitable radioactive labelsinclude, e.g., ³²P, ³³P, ¹⁴C, ¹²⁵I, ¹³¹I, ³⁵S, and ³H. Suitablefluorescent labels include, without limitation, fluorescein, fluoresceinisothiocyanate (FITC), green fluorescence protein (GFP), DyLight 488,phycoerythrin (PE), propidium iodide (PI), PerCP, PE-Alexa Fluor® 700,Cy5, allophycocyanin, and Cy7. Luminescent labels include, e.g., any ofa variety of luminescent lanthanide (e.g., europium or terbium)chelates. For example, suitable europium chelates include the europiumchelate of diethylene triamine pentaacetic acid (DTPA) ortetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA). Enzymatic labelsinclude, e.g., alkaline phosphatase, CAT, luciferase, and horseradishperoxidase. Heterologous polypeptides can be incorporated into theanti-CD200 antibodies as fusion proteins. Methods for generating nucleicacids encoding an antibody-heterologous polypeptide fusion protein arewell known in the art of antibody engineering and described in, e.g.,Dakappagari et al. (2006) J Immunol 176:426-440.

In some embodiments, the heterologous polypeptide is one that is toxicto a cell. For example, the toxic polypeptide can be selected from thegroup consisting of Pseudomonas exotoxin (PE), bryodin, gelonin,aspergillin, restrictocin, angiogenin, saporin, abrin, a prokaryoticribonuclease, a eukaryotic ribonuclease, ricin, pokeweed antiviralprotein (PAP), a pro-apoptotic polypeptide, a ribosomal inhibitoryprotein, or a biologically active fragment of any of the foregoing.Pro-apoptotic polypeptides include, e.g., Bax, Bad, Bak, Bim, Bik, Bok,Hrk, FasL, TRAIL, and TNF-α, and pro-apoptotic, biologically-activefragments thereof.

In some embodiments, an anti-CD200 antibody, an anti-CD20 antibody, orantigen-binding fragments thereof can be conjugated to a small moleculeor radioactive agent that is toxic to a cell. For example, an anti-CD200antibody or anti-CD20 antibody can be conjugated to a toxic smallmolecule selected from the group consisting of cisplatin, carboplatin,procarbazine, mechlorethamine, cyclophosphamide, calicheamicin,camptothecin, adriamycin, ifosfamide, melphalan, chlorambucil, bisulfan,nitrosurea, dactinomycin, daunorubicin, doxorubicin, bleomycin,platinum, plicomycin, monomethyl auristatin, auristatin E, mitomycin,etoposide, verampil, podophyllotoxin, tamoxifen, taxol, transplatinum,5-fluorouracil, vincristine, vinblastine, methotrexate, or an analog ofany of the aforementioned. The antibody or fragment can be conjugated toa radioactive agent that is toxic to a cell. Such radioactive agentsinclude, e.g., ⁹⁰Y, ¹⁸⁶Re, ¹⁸⁸Re, ⁶⁴Cu, ⁶⁷Cu, ²¹²Pb, ²¹²Bi, ²¹³Bi, ¹²³I,¹²⁵I, ¹³¹I, ¹¹¹In, ²¹¹At, ³²P, ¹⁷⁷Lu, ⁴⁷Sc, ¹⁰⁵Rh, ¹⁰⁹Pd, ¹⁵³Sm, or¹⁹⁹Au.

Two proteins (e.g., an anti-CD200 antibody or an anti-CD20 antibody anda heterologous moiety) can be cross-linked using any of a number ofknown chemical cross linkers. Examples of such cross linkers are thosewhich link two amino acid residues via a linkage that includes a“hindered” disulfide bond. In these linkages, a disulfide bond withinthe cross-linking unit is protected (by hindering groups on either sideof the disulfide bond) from reduction by the action, for example, ofreduced glutathione or the enzyme disulfide reductase. One suitablereagent, 4-succinimidyloxycarbonyl-α-methyl-α (2-pyridyldithio) toluene(SMPT), forms such a linkage between two proteins utilizing a terminallysine on one of the proteins and a terminal cysteine on the other.Heterobifunctional reagents that cross-link by a different couplingmoiety on each protein can also be used. Other useful cross-linkersinclude, without limitation, reagents which link two amino groups (e.g.,N-5-azido-2-nitrobenzoyloxysuccinimide), two sulfhydryl groups (e.g.,1,4-bis-maleimidobutane), an amino group and a sulfhydryl group (e.g.,m-maleimidobenzoyl-N-hydroxysuccinimide ester), an amino group and acarboxyl group (e.g., 4-[p-azidosalicylamido]butylamine), and an aminogroup and a guanidinium group that is present in the side chain ofarginine (e.g., p-azidophenyl glyoxal monohydrate).

In some embodiments, a radioactive label can be directly conjugated tothe amino acid backbone of the antibody. Alternatively, the radioactivelabel can be included as part of a larger molecule (e.g., ¹²⁵I inmeta-[¹²⁵I]iodophenyl-N-hydroxysuccinimide ([¹²⁵I]mIPNHS) which binds tofree amino groups to form meta-iodophenyl (mIP) derivatives of relevantproteins (see, e.g., Rogers et al. (1997) J Nucl Med 38:1221-1229) orchelate (e.g., to DOTA or DTPA) which is in turn bound to the proteinbackbone. Methods of conjugating the radioactive labels or largermolecules/chelates containing them to the anti-CD200 antibodies oranti-CD20 antibodies described herein are known in the art. Such methodsinvolve incubating the proteins with the radioactive label underconditions (e.g., pH, salt concentration, and/or temperature) thatfacilitate binding of the radioactive label or chelate to the protein(see, e.g., U.S. Pat. No. 6,001,329).

Methods for conjugating a fluorescent label (sometimes referred to as a“fluorophore”) to a protein (e.g., an anti-CD200 antibody, an anti-CD20antibody or antigen-binding fragments of any of the foregoing) are knownin the art of protein chemistry. For example, fluorophores can beconjugated to free amino groups (e.g., of lysines) or sulfhydryl groups(e.g., cysteines) of proteins using succinimidyl (NHS) ester ortetrafluorophenyl (TFP) ester moieties attached to the fluorophores. Insome embodiments, the fluorophores can be conjugated to aheterobifunctional cross-linker moiety such as sulfo-SMCC. Suitableconjugation methods involve incubating an antibody protein, or fragmentthereof, with the fluorophore under conditions that facilitate bindingof the fluorophore to the protein. See, e.g., Welch and Redvanly (2003)“Handbook of Radiopharmaceuticals: Radiochemistry and Applications,”John Wiley and Sons (ISBN 0471495603).

In some embodiments, the anti-CD200 antibodies or anti-CD20 antibodiescan be modified, e.g., with a moiety that improves the stabilizationand/or retention of the antibodies in circulation, e.g., in blood,serum, or other tissues. For example, the antibody or fragment can bePEGylated as described in, e.g., Lee et al. (1999) Bioconjug Chem 10(6):973-8; Kinstler et al. (2002) Advanced Drug Deliveries Reviews54:477-485; and Roberts et al. (2002) Advanced Drug Delivery Reviews54:459-476. The stabilization moiety can improve the stability, orretention of, the antibody (or fragment) by at least 1.5 (e.g., at least2, 5, 10, 15, 20, 25, 30, 40, or 50 or more) fold.

In some embodiments, the anti-CD200 antibodies, or antigen-bindingfragments thereof, described herein can be glycosylated. In someembodiments, an antibody or antigen-binding fragment thereof describedherein can be subjected to enzymatic or chemical treatment, or producedfrom a cell, such that the antibody or fragment has reduced or absentglycosylation. Methods for producing antibodies with reducedglycosylation are known in the art and described in, e.g., U.S. Pat. No.6,933,368; Wright et al. (1991) EMBO J 10(10):2717-2723; and Co et al.(1993) Mol Immunol 30:1361.

Biological Samples and Sample Collection

Suitable biological samples for use in the methods described hereininclude any biological fluid, population of cells, or tissue or fractionthereof, which includes one or more white blood cells and/or one or morered blood cells. A biological sample can be, for example, a specimenobtained from a subject (e.g., a mammal such as a human) or can bederived from such a subject. For example, a sample can be a tissuesection obtained by biopsy, or cells that are placed in or adapted totissue culture. A biological sample can also be a biological fluid suchas urine, whole blood or a fraction thereof (e.g., plasma), saliva,semen, sputum, cerebral spinal fluid, tears, or mucus. A biologicalsample can be further fractionated, if desired, to a fraction containingparticular cell types. For example, a whole blood sample can befractionated into serum or into fractions containing particular types ofblood cells such as red blood cells or white blood cells (leukocytes).If desired, a biological sample can be a combination of differentbiological samples from a subject such as a combination of a tissue andfluid sample.

The biological samples can be obtained from a subject, e.g., a subjecthaving, suspected of having, or at risk of developing, a cancer (e.g., acancer that expresses one or both of CD200 and CD20), e.g., a B-CLL. Anysuitable methods for obtaining the biological samples can be employed,although exemplary methods include, e.g., phlebotomy, swab (e.g., buccalswab), lavage, or fine needle aspirate biopsy procedure. Non-limitingexamples of tissues susceptible to fine needle aspiration include lymphnode, lung, thyroid, breast, and liver. Biological samples can also beobtained from bone marrow. Samples can also be collected, e.g., bymicrodissection (e.g., laser capture microdissection (LCM) or lasermicrodissection (LMD)), bladder wash, smear (PAP smear), or ductallavage.

Methods for obtaining and/or storing samples that preserve the activityor integrity of cells in the biological sample are well known to thoseskilled in the art. For example, a biological sample can be furthercontacted with one or more additional agents such as appropriate buffersand/or inhibitors, including protease inhibitors, the agents meant topreserve or minimize changes in the cells (e.g., changes in osmolarityor pH) or denaturation of cell surface proteins (e.g., GPI-linkedproteins) or GPI moieties on the surface of the cells. Such inhibitorsinclude, for example, chelators such as ethylenediamine tetraacetic acid(EDTA), ethylene glycol tetraacetic acid (EGTA), protease inhibitorssuch as phenylmethylsulfonyl fluoride (PMSF), aprotinin, and leupeptin.Appropriate buffers and conditions for storing or otherwise manipulatingwhole cells are described in, e.g., Pollard and Walker (1997), “BasicCell Culture Protocols,” volume 75 of Methods in molecular biology,Humana Press; Masters (2000) “Animal cell culture: a practicalapproach,” volume 232 of Practical approach series, Oxford UniversityPress; and Jones (1996) “Human cell culture protocols,” volume 2 ofMethods in molecular medicine, Humana Press.

A sample also can be processed to eliminate or minimize the presence ofinterfering substances. For example, a biological sample can befractionated or purified to remove one or more materials (e.g., cells)that are not of interest. Methods of fractionating or purifying abiological sample include, but are not limited to, flow cytometry,fluorescence activated cell sorting, and sedimentation.

Applications

The compositions described herein can be used in a number of therapeuticand diagnostic applications, e.g., the anti-CD200 antibodies describedherein can be used in methods for treating or diagnosing cancer orautoimmune disorders. For example, after it is determined that a patientis afflicted with a cancer that is resistant to treatment with ananti-CD20 therapeutic agent (e.g., an anti-CD20 antibody such asrituximab), a medical practitioner may elect to administer to the humanthe anti-CD200 antibody in an amount and with a frequency sufficient totreat the patient's cancer. In some embodiments, a medical practitionermay administer to a patient afflicted with a cancer (e.g., a liquidtumor) an anti-CD200 antibody and an anti-CD20 therapeutic agent totreat the cancer, after it has been determined that the patient's cancercomprises cancer cells expressing CD5. Methods for therapeuticallyadministering an anti-CD200 antibody to a human are well known in theart and described in, e.g., U.S. Pat. No. 7,408,041.

Methods for Treating Autoimmune Disorders

The disclosure also provides therapeutic and diagnostic applications fortreating autoimmune disorders, e.g., by reducing the concentration ofautoimmune disease-associated autoantibodies in a subject afflicted withthe disorder. For example, a medical practitioner may elect toadminister to a human with an autoimmune disorder (e.g., autoimmunehemolytic disease) an anti-CD200 antibody in an amount and with afrequency sufficient to reduce the expression (or production) of thedisorder-associated autoantibody, or to reduce the concentration of theautoantibody in the blood of the patient, to thereby treat the patient'sautoimmune disorder. Methods for therapeutically administering ananti-CD200 antibody to a human are well known in the art and describedin, e.g., U.S. Pat. No. 7,408,041.

An “autoimmune disorder,” as used herein, refers to a disease state inwhich, via the action of white blood cells (e.g., B cells, T cells,macrophages, monocytes, or dendritic cells), a pathological immuneresponse (e.g., pathological in duration and/or magnitude) has beengenerated in a host organism against a substance or a tissue that isnormally present within the host organism. Types of autoimmune diseasesinclude, but are not limited to, chronic obstructive pulmonary disease,diabetes mellitus type 1, Goodpasture's syndrome, Grave's disease,Guillain-Barré syndrome, IgA nephropathy, scleroderma, Sjögren'ssyndrome, Wegener's granulomatosis, pemphigus vulgaris, rheumatoidarthritis, Crohn's disease, Hashimoto's disease, idiopathicthrombocytopenic purpura, myasthenia gravis (MG), pulmonary biliarycirrhosis, and Miller Fisher syndrome. Autoimmune disorders also includecertain autoimmune hemolytic disorders such as antiphospholipid syndrome(APS), catastrophic anti-phospholipid syndrome (CAPS), typical oratypical hemolytic uremic syndrome (HUS), and autoimmune hemolyticanemia (AIHA). AIHA refers to a family of related diseases that arecharacterized by production of autoantibodies to host red blood cells.AIHA includes, e.g., warm AIHA (WAIHA), cold AIHA (CAD), paroxysmal coldhemoglobinuria (PCH), and drug-induced hemolytic anemias (DIHA).

A human “at risk of developing an autoimmune disorder” refers to a humanwith a family history of autoimmune disorders (e.g., a geneticpredisposition to one or more inflammatory disorders) or one exposed toone or more autoimmune disorder/autoantibody-inducing conditions. Forexample, a human exposed to a shiga toxin is at risk for developingtypical HUS. Humans with certain cancers (e.g., liquid tumors such asmultiple myeloma or chronic lymphocytic leukemia) can pre-disposepatients to developing certain autoimmune hemolytic diseases. Forexample, PCH can follow a variety of infections (e.g., syphilis) orneoplasms such as non-Hodgkin's lymphoma. In another example, CAD can beassociated with HIV infection, Mycoplasma pneumonia infection,non-Hodgkin's lymphoma, or Waldenstrom's macroglobulinemia. In yetanother example, autoimmune hemolytic anemia is a well-knowncomplication of human chronic lymphocytic leukemia, approximately 11% ofCLL patients with advanced disease will develop AIHA. As many as 30% ofCLL may be at risk for developing AIHA. See, e.g., Diehl et al. (1998)Semin Oncol 25(1):80-97 and Gupta et al. (2002) Leukemia16(10):2092-2095. From the above it will be clear that humans “at riskof developing an autoimmune disorder” are not all the humans within aspecies of interest.

A human “suspected of having an autoimmune disorder” is one who presentswith one or more symptoms of an autoimmune disorder. Symptoms ofautoimmune disorders can vary in severity and type with the particularautoimmune disorder and include, but are not limited to, redness,swelling (e.g., swollen joints), joints that are warm to the touch,joint pain, stiffness, loss of joint function, fever, chills, fatigue,loss of energy, pain, fever, pallor, icterus, urticarial dermaleruption, hemoglobinuria, hemoglobinemia, and anemia (e.g., severeanemia), headaches, loss of appetite, muscle stiffness, insomnia,itchiness, stuffy nose, sneezing, coughing, one or more neurologicsymptoms such as dizziness, seizures, or pain. From the above it will beclear that not all humans are “suspected of having an autoimmunedisorder.”

In some embodiments, the medical practitioner can administer ananti-CD200 antibody to a human in an amount effective to reduce theexpression or production of an autoimmune disorder-associatedautoantibody in the human. For example, PCH most commonly results fromthe production by a subject of an autoantibody (known as the“Donath-Landsteiner antibody”) that binds to the P antigen of red bloodcells in cold temperatures. Once bound to the red blood cells, theantibody facilitates complement-mediated hemolysis of the cells atwarmer temperatures. As many as 40% of immune hemolytic anemias inchildren are associated with the Donath-Landsteiner antibody. See, e.g.,Sokol et al. (1982) Acta Haematol 68(4):268-77. Thus, e.g., ananti-CD200 antibody can be administered to a PCH patient in an amountsufficient to reduce the production or expression of theDonath-Landsteiner antibody in the human to thereby treat the human'sPCH.

Similarly, CAD (or cold hemagglutinin disease or CHD/CHAD) is anautoimmune disorder characterized by autoantibodies that bind to the Iantigen on red blood cells at colder temperatures. Once bound, theantibodies facilitate hemagglutination, and complement-mediatedhemolysis, of the cells. Thus, a medical practitioner can administer ananti-CD200 antibody in an amount sufficient to reduce the production orexpression of the anti-I antigen antibodies in the human to therebytreat the human's CAD.

In another example, a large number of patients with MG expressantibodies that bind to and inhibit the activity of the nicotinicacetylcholine receptor (AChR). The antibodies cause loss ofacetylcholine receptors and diminished receptor function at the muscleend-plate of the mature neuromuscular junction. Some patients who arelacking in detectable anti-AChR antibodies instead expressauto-antibodies directed to a muscle-specific receptor tyrosine kinase(MuSK). See, e.g., Hoch et al. (2001) Nature Med. 7(3):365-368). Thus, amedical practitioner can administer an anti-CD200 antibody in an amountsufficient to reduce the production or expression of the anti-AChR oranti-MuSK antibodies in the human to thereby treat the human's MG.

Methods for detecting the presence or amount of an autoimmunedisorder-associated autoantibody in a human are well known in the artand are described in, e.g., Burbelo et al. (2009) J Transl Med 7:83;Hanke et al. (2009) Arthritis Res Ther 11(1):R22; Hoch et al. (2001),supra; Vernino et al. (2008) J Neuroimmunol 197(1):63-69; Sokol et al.(1982), supra; and Littleton et al. (2009) Mol Cell Proteomics8(7):1688-1696.

In some embodiments, the anti-CD200 antibody is administered to asubject in an amount and with a frequency to maintain a reducedconcentration (or a reduced expression or production) of the autoimmunedisorder-associated autoantibody. Methods for detecting expression or achange in concentration of autoantibodies are well known in the art(e.g., Western blot, immunohistochemistry, and flow cytometrytechniques) and described herein. Through an iterative process, amedical practitioner can determine the appropriate dose amount, andfrequency of administration of each dose, required to maintain a reducedconcentration of the autoimmune disorder-associated autoantibodies inthe patient. For example, a medical practitioner can administer to apatient with an autoimmune disorder such as AIHA one or more (e.g., one,two, three, four, five, six, seven, eight, nine, or 10 or more or, e.g.,at least two, at least three, four, five, six, seven, or eight or more)times an anti-CD200 antibody in an amount that reduces (or is at leastexpected to reduce) the concentration of autoantibodies in the human.The at least two doses should be spaced apart in time by at least one(e.g., at least two, three, four, five, six, seven, eight, nine, 10, 11,12, 13, or even 14) day(s). Biological samples (e.g., blood samples)containing the autoantibodies are obtained from the patient at varioustimes, e.g., prior to the first anti-CD200 antibody administration,between the first dose and at least one additional dose, and at leastone biological sample collection following the second dose. In someembodiments, biological samples may be collected at least two timesbetween doses and/or at least one time after the final dose administeredto the patient. The autoantibodies in each biological sample obtainedare then interrogated for relative titer of the autoimmune-diseaseassociated autoantibody to determine whether the amount and/or thefrequency of administration of the anti-CD200 antibody are sufficient tomaintain a reduced concentration of the autoantibody in the patient. Themedical practitioner (and/or a computer) can determine an anti-CD200antibody dosing schedule for the patient that is sufficient to maintaina reduced concentration of autoimmune disorder-associated autoantibodiesin the patient over the course of the treatment.

In some embodiments, administration of the anti-CD200 antibody to thehuman reduces the autoantibody concentration by at least 5 (e.g., atleast 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30,35, 40, 45, 50, 55, 60, 65, 70, 75, 80, or 85 or more) %.

In some embodiments, the anti-CD200 antibody can be chronicallyadministered to the human. As used herein, “chronically administered,”“chronic treatment,” “treating chronically,” or similar grammaticalvariations thereof refer to a treatment regimen that is employed tomaintain a certain threshold concentration of a therapeutic agent in theblood of a patient in order to maintain a particular state in thepatient over a prolonged period of time. For example, an anti-CD200antibody can be chronically administered a patient with MG to maintain areduced concentration of anti-AChR antibodies in the blood of thepatient for a prolonged period of time. Accordingly, a patientchronically treated with a anti-CD200 antibody can be treated for aperiod of time that is greater than or equal to 2 weeks (e.g., 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 44, 45, 46, 47, 48, 49, 50, 51, or 52 weeks; 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, or 12 months; or 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6,6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, or 12 years or for the remainderof the patient's life).

The inventors have identified and provided herein several biomarkersconsistent with the production in a human of an immunomodulatory effectby an anti-CD200 antibody administered to the human. That is, uponadministration of the antibody to mice with an autoimmune disease(autoimmune hemolytic disease), the concentration of a number ofsplenocyte and bone marrow cell subsets changed in the mice. An“immunomodulatory effect” and grammatically similar terms, as usedherein, refer to a measurable immunological effect in an animal (e.g., ahuman) attributable to the biological activity of an anti-CD200 antibodyadministered to an animal (e.g., a human). For example, the inventorshave observed that following administration of an anti-CD200 antibody toa mouse, the concentration of the following CD200⁺ leukocyte populationsis reduced: CD3⁺/CD200⁺ cells, CD45R⁺/CD200⁺ cells, CD5⁺/CD200⁺ cells,CD19⁺/CD200⁺ cells, CD138⁺/CD200⁺ cells, CD45R⁺/CD138⁺/CD200⁺, andCD200R⁺/CD200⁺ cells. In some embodiments, the CD200⁺ leukocytes arelocalized in the spleen. The reduction of the aforementioned CD200⁺ cellsubsets can also be observed in peripheral blood. Also observed was thatupon administration of an anti-CD200 antibody to a mouse, theconcentration of the following CD200⁺ bone marrow cell subsets isreduced: CD200⁺ bone marrow cells, Igk⁺/CD200⁺ bone marrow cells,CD45R⁺/CD138⁺/CD200⁺ bone marrow cells, CD138⁺/CD200⁺ bone marrow cells,c-kit⁺/CD200⁺ bone marrow cells, and c-kit⁺/CD200⁺/Lin⁻ bone marrowcells. While not being bound by any particular theory or mechanism ofaction, the inventors believe that monitoring a patient treated with ananti-CD200 antibody for the occurrence of one or more of thesebiomarkers is useful for, at bottom, determining whether the anti-CD200antibody is capable of producing a biological effect in the human towhom the antibody is administered. Moreover, one or more of thebiomarkers are also useful for identifying a dose—a threshold dose—of ananti-CD200 antibody, such as samalizumab (ALXN6000), that by virtue ofits immunomodulatory effect in the human is sufficient to achieve aclinically-meaningful effect in the disease (i.e., sufficient to treat adisease such as an autoimmune disorder or a cancer). To wit, mice withautoimmune hemolytic disease treated with an anti-CD200 antibodyexhibited a reduced concentration of the disease-associated autoantibodyin the mice.

Thus, in accordance with the present disclosure, a medical practitionercan administer to a human in need thereof an anti-CD200 antibody in anamount and with a frequency sufficient to treat the autoimmune disorderby maintaining one or more of the following physiological conditions(immunological effects) in the human: (i) a decreased concentration ofat least one CD200⁺ leukocyte subset (e.g., at least one CD200⁺splenocyte subset); (ii) an increased concentration of splenic orperipheral F4/80⁺ cells; and (iii) a decreased concentration of at leastone bone marrow stem cell subset. The CD200⁺ leukocyte subsets can be,e.g., CD3⁺/CD200⁺ cells, CD45R⁺/CD200⁺ cells, CD5⁺/CD200⁺ cells,CD19⁺/CD200⁺ cells, CD138⁺/CD200⁺ cells, CD45R⁺/CD138⁺/CD200⁺, andCD200R⁺/CD200⁺ cells. The CD200⁺ bone marrow cell subsets can be, e.g.,CD200⁺ bone marrow cells, Igk⁺/CD200⁺ bone marrow cells,CD45R⁺/CD138⁺/CD200⁺ bone marrow cells, CD138⁺/CD200⁺ bone marrow cells,c-kit⁺/CD200⁺ bone marrow cells, and c-kit⁺/CD200⁺/Lin⁻ bone marrowcells. The splenic or peripheral F4/80⁺ cells can be macrophages. Insome cases, at least two of the physiological conditions are maintained.In some embodiments, all of the conditions are maintained in the humanthroughout the treatment period.

Methods for measuring the concentration of CD200⁺ cells (e.g., any ofthe CD200⁺ leukocyte or bone marrow cell subsets) are well known in theart and include, among other methods, flow cytometry. See, e.g., Chen etal. (2009) Mol Immunol 46(10):1951-1963. A suitable method for detectingand/or measuring the concentration of CD200⁺ bone marrow cell,splenocyte, or peripheral blood leukocyte subsets is also set forth inthe working examples. In some embodiments, a practitioner caninterrogate a biological sample obtained from a post-treatment patient(a patient to which an anti-CD200 antibody has been administered) forthe concentration of cells of a particular subset of CD200⁺ cells. Forexample, a practitioner can determine the concentration of CD45R⁺/CD200⁺leukocytes and/or the concentration of c-kit⁺/CD200⁺/Lin⁻ bone marrowcells present in a biological sample from a post-treatment patient.

In some embodiments, following administration of an anti-CD200 antibodyto a human the concentration of a CD200⁺ leukocyte (e.g., CD200⁺leukocyte population in spleen) or bone marrow cell subset that is atleast 5% less than the concentration of the corresponding subset in thehuman prior to the treatment. In some embodiments, a post-treatmentCD200⁺ splenocyte or bone marrow cell subset concentration that is atleast 10 (e.g., 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 44, 45, 50, 55, 60, 65, 70, 75, 80, or more than 80) % less than theconcentration of the corresponding subset prior to treatment with theantibody.

An anti-CD200 antibody described herein can be co-administered with oneor more additional therapeutic agents useful for treating or preventingan inflammatory condition. The one or more agents include, e.g., anon-steroidal anti-inflammatory drug (NSAID), a disease-modifyinganti-rheumatic drug (DMARD), a biological response modifier, or acorticosteroid. Biological response modifiers include, e.g., an anti-TNFagent (e.g., a soluble TNF receptor or an antibody specific for TNF suchas adulimumab, infliximab, or etanercept). In some embodiments, the oneor more additional therapeutic agents can be, e.g., steroids,anti-malarials, aspirin, non-steroidal anti-inflammatory drugs,immunosuppressants, cytotoxic drugs, corticosteroids (e.g., prednisone,dexamethasone, and prednisolone), methotrexate, methylprednisolone,macrolide immunosuppressants (e.g., sirolimus and tacrolimus), mitoticinhibitors (e.g., azathioprine, cyclophosphamide, and methotrexate),fungal metabolites that inhibit the activity of T lymphocytes (e.g.,cyclosporine), mycophenolate mofetil, glatiramer acetate, and cytotoxicand DNA-damaging agents (e.g., chlorambucil or any other DNA-damagingagent described herein or known in the art).

Methods for Treating Cancers

The disclosure also provides therapeutic and diagnostic applications fortreating cancers. For example, after it is determined that a human has atumor that comprises tumor cells expressing CD200, a medicalpractitioner may elect to administer to the human the anti-CD200antibody in an amount and with a frequency sufficient to treat thepatient's cancer. Methods for therapeutically administering ananti-CD200 antibody to a human are well known in the art and describedin, e.g., U.S. Pat. No. 7,408,041.

Cancer is a class of diseases or disorders characterized by uncontrolleddivision of cells and the ability of these to spread, either by directgrowth into adjacent tissue through invasion, or by implantation intodistant sites by metastasis (where cancer cells are transported throughthe bloodstream or lymphatic system). Cancer can affect people at allages, but risk tends to increase with age. Types of cancers can include,e.g., lung cancer, breast cancer, colon cancer, pancreatic cancer, renalcancer, stomach cancer, liver cancer, bone cancer, hematological cancer,neural tissue cancer (e.g., neuroblastoma), melanoma, thyroid cancer,ovarian cancer, a liquid tumor, testicular cancer, prostate cancer,cervical cancer, vaginal cancer, or bladder cancer. Liquid tumorsinclude, e.g., leukemias (e.g., chronic lymphocytic leukemia such as Bcell or T cell type chronic lymphocytic leukemia) and multiple myeloma.Bone cancers include, without limitation, osteosarcoma andosteocarcinomas.

As used herein, a human “at risk of developing a cancer” is a human thathas a predisposition to develop a cancer, i.e., a genetic predispositionto develop cancer such as a mutation in a tumor suppressor gene (e.g.,mutation in BRCA1, p53, RB, or APC or other genetic rearrangements) orhas been exposed to conditions that can result in cancer. Thus, a humancan also be one “at risk of developing a cancer” when the human has beenexposed to mutagenic or carcinogenic levels of certain compounds (e.g.,carcinogenic compounds in cigarette smoke such as acrolein, arsenic,benzene, benz {a} anthracene, benzo{a}pyrene, polonium-210 (radon),urethane, or vinyl chloride). Moreover, the human can be “at risk ofdeveloping a cancer” when the human has been exposed to, e.g., largedoses of ultraviolet light or X-irradiation, or infected by atumor-causing/associated virus such as a papillomavirus, Epstein-Barrvirus, hepatitis B virus, or human T-cell leukemia-lymphoma virus. Fromthe above it will be clear that not all humans are “at risk ofdeveloping a cancer.”

A human “suspected of having a cancer” is one having one or moresymptoms of a cancer. Symptoms of cancer are well-known to those ofskill in the art and include, without limitation, breast lumps, pain,weight loss, weakness, excessive fatigue, difficulty eating, loss ofappetite, chronic cough, worsening breathlessness, coughing up blood,blood in the urine, blood in stool, nausea, vomiting, liver metastases,lung metastases, bone metastases, abdominal fullness, bloating, fluid inperitoneal cavity, vaginal bleeding, constipation, abdominal distension,perforation of colon, acute peritonitis (infection, fever, pain), pain,vomiting blood, heavy sweating, fever, high blood pressure, anemia,diarrhea, jaundice, dizziness, chills, muscle spasms, and difficultyswallowing. Symptoms of a primary cancer (e.g., a large primary cancer)can include, e.g., any one of colon metastases, lung metastases, bladdermetastases, liver metastases, bone metastases, kidney metastases, andpancreas metastases.

The inventors have discovered that administration of an anti-CD200antibody to an animal resulted in a marked reduction in theconcentration of CD5⁺ cells in the spleen of the animal. While thedisclosure is not bound by any particular theory or mechanism of action,it is likely that CD5⁺ CLL cells may be refractory to rituximab therapyat least in part because of a reduced expression by the cells of CD20.The inventors have shown that a therapeutic composition containing ananti-CD200 antibody is useful for reducing CD5⁺ cell populations in ananimal and thus believe that the composition is particularly useful fortreating a subset of CLL patients that are refractory to treatment withanti-CD20 therapy (e.g., rituximab-resistant).

Accordingly, the disclosure features a variety of methods for treatingcancers, particularly for selecting or identifying which cancers maymost benefit from treatment with an anti-CD200 antibody. For example,the disclosure features a method for treating a human afflicted withcancer that is resistant, suspected to be resistant, or likely to beresistant, to treatment with an anti-CD20 therapeutic agent such asrituximab. “Resistance” to a therapy, “refractory” to therapy, and likegrammatical phrases, as used herein, refer to a patient's clinical stateof being, in which there is a reduction in the effectiveness of a giventreatment (e.g., treatment with an anti-CD20 therapeutic agent) intreating or curing a given disorder (e.g., a cancer) or a reduction inthe effectiveness of the treatment in ameliorating one or more symptomsassociated with the disorder. For example, the therapeutic benefits ofan anti-CD20 therapy to a patient afflicted with a liquid tumor such asB cell chronic lymphocytic leukemia may diminish over time such that thecancer recurs, remains, or progresses even in the presence of thetherapy. Resistance by cancers to therapeutic agents such as anti-CD20therapeutic agents is well-documented in the art of medicine and isdescribed in, e.g., Reddy et al. (2006) J Clin Oncol 24(18S):17509;Bello and Sotomayor (2007) Hematology 1:233; Hernandez-Ilizaliturri etal. (2009) J Clin Oncol 27(15s):8543; and Friedberg et al. (2005)Hematology 1:329.

In some embodiments, a patient can have a cancer that is suspected ofbeing resistant or is likely to become resistant to an anti-CD20therapy. One biomarker useful in assessing whether a cancer is likely tobecome resistant to an anti-CD20 therapeutic agent such as rituximab isthe presence or concentration of CD5⁺ cancer cells in the population. Asdescribed above, because the CD5⁺ cells express reduced levels of CD20,the cells are less affected by the anti-CD20 therapy and thus can beselected for due to a growth advantage over cancer cells that expresshigher levels of CD20. Methods for detecting the expression of CD5 arewell known in the art of molecular biology and include, withoutlimitation, Western blotting, dot blotting, and flow cytometry, whichare useful for quantifying expression of CD5 protein, or reversetranscriptase polymerase chain reaction (RT-PCR) and Northern blottinganalysis for quantifying expression of CD5 mRNA. See, e.g., Ennishi etal. (2008), supra; Holodick et al. (2009), supra; Garaud et al. (2009) JImmunol 182(9):5623-5632; and McNab et al. (2009) Ann Clin Lab Sci39(2):108-113. See generally Sambrook et al. (1989) “Molecular Cloning:A Laboratory Manual, 2^(nd) Edition,” Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y. and Ausubel et al. (1992) “CurrentProtocols in Molecular Biology,” Greene Publishing Associates. Asuitable method for detecting and/or quantifying the expression of CD5by cells, or for determining the percentages of CD5 expressing cells ina population, is flow cytometry and is exemplified in the workingexamples.

In some embodiments, a cancer that is likely to be resistant to ananti-CD20 therapeutic agent comprises at least a plurality or a portion(e.g., two or more; at least 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.5%,2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 15%, 17%, 20%, 25%, 30%, 35%,40%, or 45% or more) of cancer cells (e.g., B cell chronic lymphocyticleukemia cells) expressing CD5. In some embodiments, greater than 45(e.g., greater than 50, 55, 60, 65, 70, 75, or 80 or more) % of apatient's cancer cells can express CD5. In some embodiments, the cancercomprises cells (e.g., a plurality or even a majority of cells) thatexpress or overexpress CD5 (e.g., CD5 protein and/or CD5 mRNA). In someembodiments, at least (or greater than) 10 (e.g., 15, 20, 25, 30, 35,40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95) % of the cancer cellsof the human's cancer overexpress CD5. In some embodiments, all assayedcancer cells overexpress CD5 relative to normal cells. In someembodiments, a cancer cell (e.g., a plurality of cancer cells, at least10% of cancer cells, or all assayed cancer cells) can express CD5protein at levels at least about 1.4 (e.g., at least about 1.5, 1.6,1.7, 1.8, 1.9, 2.0, 2.2, 2.5, 3.0, 3.5, 4.0, 4.5, or 5 or more)-foldhigher than the expression levels found on normal cells of the samehistological type or higher than the average expression of normal cellsfrom one or more patients who do not have cancer.

In some embodiments, the methods described herein can includedetermining whether the human has a cancer. In some embodiments, themethods described herein can include the step of determining whether oneor more cancer cells of a human's cancer express CD200. In someembodiments, the methods can include determining whether one or morecancer cells of the human's cancer overexpress CD200, relative to acontrol sample. In some embodiments, the control sample is obtained fromthe same human and comprises normal cells of the same tissue type as thehuman's cancer. For example, a skilled artisan could measure the levelof CD200 protein present on colon cancer cells from a patient ascompared to normal colon cells from the patient. In some embodiments,the control sample can be the expression level (or average expressionlevel) of cells obtained from one or more humans who do not have cancer.In some embodiments, the cancer comprises cells (e.g., a plurality oreven a majority of cells) that express or overexpress CD200 (e.g., CD200protein and/or CD200 mRNA). In some embodiments, at least (or greaterthan) 10 (e.g., 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80,85, 90, or 95) % of the cancer cells of the human's cancer overexpressCD200. In some embodiments, all assayed cancer cells overexpress CD200relative to normal cells. In some embodiments, a cancer cell (e.g., aplurality of cancer cells, at least 10% of cancer cells, or all assayedcancer cells) can express CD200 protein at levels at least about 1.4(e.g., at least about 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.2, 2.5, 3.0, 3.5,4.0, 4.5, or 5 or more)-fold higher than the expression levels found onnormal cells of the same histological type or higher than the averageexpression of normal cells from one or more patients who do not havecancer.

In some embodiments, an anti-CD200 antibody is only administered to ahuman if the human's cancer expresses or overexpresses CD200. Methodsfor detecting expression of CD200 are well known in the art and include,e.g., Western blot, immunohistochemistry, and flow cytometry techniques.Suitable methods for detecting CD200 expression are described in detailin, e.g., Kretz-Rommel et al. (2007) J Immunol 178:5595-5605 andKretz-Rommel et al. (2008) J Immunol 180:699-705. In some embodiments,an anti-CD200 antibody is only administered to a human if the human'scancer expresses or overexpresses CD200 and CD5.

In some embodiments, an anti-CD200 antibody blocks immune suppression incancer by targeting cancer cells that express CD200. Eradication, orinhibition, of these cancer cells can stimulate the immune system andallow further eradication of cancer cells.

In some embodiments, the combination of direct cancer cell killing anddriving the immune response towards a Th1 profile provides enhancedefficacy in cancer treatment. Thus, in one embodiment, a cancertreatment is provided wherein an antibody or antibody fragment, whichbinds to CD200 and both a) blocks the interaction between CD200 and itsreceptor and b) directly kills the cancer cells expressing CD200, isadministered to a cancer patient. The mechanisms by which the cancercells are killed can include, but are not limited to, ADCC or CDC;fusion with a toxin; fusion with a toxic radioactive agent; fusion witha toxic polypeptide such as granzyme B or perforin; fusion with acytotoxic virus (e.g., cytotoxic reovirus such as Reolysin®); or fusionwith a cytokine such as TNF-α or IFN-α. In an alternative embodiment, acancer treatment involves administering an antibody that both a) blocksthe interaction between CD200 and its receptor and b) enhances cytotoxicT cell or NK cell activity against the tumor. Such enhancement of thecytotoxic T cell or NK cell activity may, for example, be combined byfusing the antibody with cytokines such as, e.g., IL-2, IL-12, IL-18,IL-13, and IL-5. In addition, such enhancement may be achieved byadministration of an anti-CD200 antibody in combination with inhibitorssuch as IMiDs, thalidomide, or thalidomide analogs.

In yet another embodiment, the cancer treatment involves administeringan antibody that both a) blocks the interaction between CD200 and itsreceptor and b) attracts T cells to the tumor cells. T cell attractioncan be achieved by fusing the Ab with chemokines such as MIG, IP-10,I-TAC, CCL21, CCL5 or LIGHT. Also, treatment with chemotherapeutics canresult in the desired upregulation of LIGHT. The combined action ofblocking immune suppression and killing directly through antibodytargeting of the tumor cells is a unique approach that providesincreased efficacy.

The disclosure also provides a method of treating a cancer using acombination therapy of an anti-CD200 antibody and an anti-CD20therapeutic agent. While not being bound by any particular theory ormechanism of action, the inventors believe that administration of ananti-CD200 antibody will enhance the efficacy of an anti-CD20therapeutic agent by reducing the proportion of cancer cells that arelikely to be refractory, namely the CD5⁺ cancer cells. The anti-CD20therapeutic agent can be any of those described herein or known in theart such as, e.g., rituximab (Biogen Idec), ⁹⁰Y-ibritumomab tiuxetan(Biogen Idec), ¹³¹I-tositumomab (GlaxoSmithKline), ofatumumab (Genmab),TRU-015 (Trubion), veltuzumab (IMMU-106; Immunomedics), ocrelizumab(Roche), and AME-133v (Applied Molecular Evolution).

In some embodiments, the anti-CD200 antibody and the anti-CD20therapeutic agent are separate agents. Accordingly, the anti-CD200antibody and the anti-CD20 therapeutic agent can be administered at thesame time. In other embodiments, the anti-CD200 antibody is administeredfirst in time and the anti-CD20 therapeutic agent is administered secondin time. In some embodiments, the anti-CD20 therapy is administeredfirst in time and the anti-CD200 antibody is administered second intime.

The anti-CD200 antibody can replace or augment a previously or currentlyadministered therapy such as an anti-CD20 therapeutic agent. Forexample, upon treating with an anti-CD200 antibody or antigen-bindingfragment thereof, administration of the anti-CD20 therapeutic agent cancease or diminish, e.g., be administered at lower levels. In someembodiments, administration of the anti-CD20 therapeutic agent can bemaintained. In some embodiments, administration of the anti-CD20therapeutic agent will be maintained until the level of the anti-CD200antibody reaches a level sufficient to provide a therapeutic effect. Thetwo therapies can be administered in combination as a single agent,e.g., a bispecific antibody that binds to both CD200 and CD20 (seeabove).

An anti-CD200 antibody described herein can also be co-administered to ahuman with cancer along with one or more additional therapeuticanti-cancer agents. Likewise an anti-CD200 antibody described herein canbe co-administered to a human with cancer along with an anti-CD20therapeutic agent and one or more additional therapeutic anti-canceragents. Anti-cancer agents include, e.g., chemotherapeutic agents,ionizing radiation, immunotherapy agents, or hyperthermotherapy agents.Chemotherapeutic agents include, but are not limited to,aminoglutethimide, amsacrine, anastrozole, asparaginase, bcg,bicalutamide, bleomycin, buserelin, busulfan, camptothecin,capecitabine, carboplatin, carmustine, chlorambucil, cisplatin,cladribine, clodronate, colchicine, cyclophosphamide, cyproterone,cytarabine, dacarbazine, dactinomycin, daunorubicin, dienestrol,diethylstilbestrol, docetaxel, doxorubicin, epirubicin, estradiol,estramustine, etoposide, exemestane, filgrastim, fludarabine,fludrocortisone, fluorouracil, fluoxymesterone, flutamide, gemcitabine,genistein, goserelin, hydroxyurea, idarubicin, ifosfamide, imatinib,interferon, irinotecan, letrozole, leucovorin, leuprolide, levamisole,lomustine, mechlorethamine, medroxyprogesterone, megestrol, melphalan,mercaptopurine, mesna, methotrexate, mitomycin, mitotane, mitoxantrone,nilutamide, nocodazole, octreotide, oxaliplatin, paclitaxel,pamidronate, pentostatin, plicamycin, porfimer, procarbazine,raltitrexed, rituximab, streptozocin, suramin, tamoxifen, taxol,temozolomide, teniposide, testosterone, thioguanine, thiotepa,titanocene dichloride, topotecan, trastuzumab, tretinoin, vinblastine,vincristine, vindesine, and vinorelbine. In some embodiments, apharmaceutical composition comprising an anti-CD200 antibody orCD200-binding fragment thereof can be co-formulated with one or more ofany of the foregoing agents or any other anti-cancer agent describedherein.

These chemotherapeutic anti-tumor compounds may be categorized by theirmechanism of action into groups, including, for example, the following:anti-metabolites/anti-cancer agents, such as pyrimidine analogs(5-fluorouracil, floxuridine, capecitabine, gemcitabine and cytarabine)and purine analogs, folate antagonists and related inhibitors(mercaptopurine, thioguanine, pentostatin and 2-chlorodeoxyadenosine(cladribine)); antiproliferative/antimitotic agents including naturalproducts such as vinca alkaloids (vinblastine, vincristine, andvinorelbine), microtubule disruptors such as taxane (paclitaxel,docetaxel), vincristine, vinblastine, nocodazole, epothilones andnavelbine, epidipodophyllotoxins (etoposide, teniposide), DNA damagingagents (actinomycin, amsacrine, anthracyclines, bleomycin, busulfan,camptothecin, carboplatin, chlorambucil, cisplatin, cyclophosphamide,cytoxan, dactinomycin, daunorubicin, doxorubicin, epirubicin,hexamethylmelamineoxaliplatin, iphosphamide, melphalan, mechlorethamine,mitomycin, mitoxantrone, nitrosourea, plicamycin, procarbazine, taxol,taxotere, teniposide, triethylenethiophosphoramide and etoposide(VP16)); antibiotics such as dactinomycin (actinomycin D), daunorubicin,doxorubicin (adriamycin), idarubicin, anthracyclines, mitoxantrone,bleomycins, plicamycin (mithramycin) and mitomycin; enzymes(L-asparaginase which systemically metabolizes L-asparagine and deprivescells which do not have the capacity to synthesize their ownasparagine); antiplatelet agents; antiproliferative/antimitoticalkylating agents such as nitrogen mustards (mechlorethamine,cyclophosphamide and analogs, melphalan, chlorambucil), ethyleniminesand methylmelamines (hexamethylmelamine and thiotepa), alkylsulfonates-busulfan, nitrosoureas (carmustine (BCNU) and analogs,streptozocin), trazenes—dacarbazinine (DTIC);antiproliferative/antimitotic antimetabolites such as folic acid analogs(methotrexate); platinum coordination complexes (cisplatin,carboplatin), procarbazine, hydroxyurea, mitotane, aminoglutethimide;hormones, hormone analogs (estrogen, tamoxifen, goserelin, bicalutamide,nilutamide) and aromatase inhibitors (letrozole, anastrozole);anticoagulants (heparin, synthetic heparin salts and other inhibitors ofthrombin); fibrinolytic agents (such as tissue plasminogen activator,streptokinase and urokinase), aspirin, dipyridamole, ticlopidine,clopidogrel, abciximab; antimigratory agents; antisecretory agents(breveldin); immunosuppressives (cyclosporine, tacrolimus (FK-506),sirolimus (rapamycin), azathioprine, mycophenolate mofetil);immunomodulatory agents (thalidomide and analogs thereof such aslenalidomide (Revlimid, CC-5013) and CC-4047 (Actimid)),cyclophosphamide; anti-angiogenic compounds (TNP-470, genistein) andgrowth factor inhibitors (vascular endothelial growth factor (VEGF)inhibitors, fibroblast growth factor (FGF) inhibitors); angiotensinreceptor blocker; nitric oxide donors; anti-sense oligonucleotides;antibodies (trastuzumab); cell cycle inhibitors and differentiationinducers (tretinoin); mTOR inhibitors, topoisomerase inhibitors(doxorubicin (adriamycin), amsacrine, camptothecin, daunorubicin,dactinomycin, eniposide, epirubicin, etoposide, idarubicin andmitoxantrone, topotecan, irinotecan), corticosteroids (cortisone,dexamethasone, hydrocortisone, methylprednisolone, prednisone, andprednisolone); growth factor signal transduction kinase inhibitors;mitochondrial dysfunction inducers and caspase activators; and chromatindisruptors.

In some embodiments, the methods described herein can include, afteradministering the anti-CD200 antibody, monitoring the human for animprovement in the disorder and/or one or more symptoms thereof.Monitoring a human for an improvement in a disorder (e.g., a cancer, aninflammatory condition, or a disorder associated with bone loss), asdefined herein, means evaluating the subject for a change in a diseaseparameter, e.g., an improvement in one or more symptoms of the disease.In some embodiments, the evaluation is performed at least 1 hour, e.g.,at least 2, 4, 6, 8, 12, 24, or 48 hours, or at least 1 day, 2 days, 4days, 10 days, 13 days, 20 days or more, or at least 1 week, 2 weeks, 4weeks, 10 weeks, 13 weeks, 20 weeks or more, after an administration.The human can be evaluated in one or more of the following periods:prior to beginning of treatment; during the treatment; or after one ormore elements of the treatment have been administered. Evaluating caninclude evaluating the need for further treatment, e.g., evaluatingwhether a dosage, frequency of administration, or duration of treatmentshould be altered. It can also include evaluating the need to add ordrop a selected therapeutic modality, e.g., adding or dropping any ofthe treatments for a disorder described herein.

In some embodiments, monitoring the progress and/or effectiveness of atherapeutic treatment includes monitoring the level of CD200 expressionby cancer cells before and after treatment. In some embodiments,monitoring the progress and/or effectiveness of a therapeutic treatmentincludes monitoring the level of CD5 expression by cancer cells beforeand after treatment. In some embodiments, monitoring the progress and/oreffectiveness of a therapeutic treatment includes monitoring the levelof CD200 and CD5 expression by cancer cells before and after treatment.For example, pre-treatment levels of CD200 and/or CD5 expression bycancer cells can be ascertained and, after at least one administrationof the therapy, levels of CD200 and/or CD5 can again be determined. Adecrease in CD200 and/or CD5 expression by cancer cells can beindicative of an effective treatment (see below). Measurement of CD200and/or CD5 expression levels by the cancer cells can be used by thepractitioner as a guide for increasing dosage amount or frequency of thetherapy. It should of course be understood that CD200 and/or CD5 levelscan be directly monitored or, alternatively, any marker that correlateswith CD200 and/or CD5 can be monitored.

Because administration of an anti-CD200 antibody to an animal reducesthe concentration of CD5⁺ cells in the animal, it is believed to bebeneficial to administer to the human an anti-CD200 antibody in anamount and with a frequency sufficient to sustain the reducedconcentration of CD5⁺ cells in a human particularly in the case of CD5⁺chronic lymphocytic leukemia for the reasons described above. Methodsfor detecting expression or a change in expression of CD5 by cells(e.g., cancer cells such as B cell CLL cells) are well known in the art(e.g., Western blot, immunohistochemistry, and flow cytometrytechniques) and described herein. For example, following theadministration of an anti-CD200 antibody to a human, the concentrationof CD5⁺ cancer cells in the human can be determined by flow cytometryanalysis of the cancer cells present in a biological sample obtainedfrom a patient. The concentration of CD5⁺ cancer cells post-treatmentcan be compared to a control concentration (e.g., the concentration ofCD5⁺ cancer cells in the human prior to treatment with the antibody),wherein a reduction in the concentration of CD5⁺ cancer cells indicatesthat the anti-CD200 antibody has been administered to the human in anamount and with a frequency sufficient to reduce the concentration ofCD5⁺ cells in the human and is thus therapeutically effective.

Through an iterative process, a medical practitioner can determine theappropriate dose amount, and frequency of administration of each dose,required to maintain a reduced concentration of CD5⁺ cancer cells in thepatient. For example, a medical practitioner can administer to a cancerpatient one or more (e.g., one, two, three, four, five, six, seven,eight, nine, or 10 or more or, e.g., at least three, four, five, six,seven, or eight or more) times an anti-CD200 antibody in an amount thatreduces (or is at least expected to reduce) the concentration of CD5⁺cancer cells. The at least two doses should be spaced apart in time byat least one (e.g., at least two, three, four, five, six, seven, eight,nine, 10, 11, 12, 13, or even 14) day(s). Biological samples (e.g.,blood samples) containing cancer cells are obtained from the patient atvarious times, e.g., prior to the first anti-CD200 antibodyadministration, between the first dose and at least one additional dose,and at least one biological sample collection following the second dose.In some embodiments, biological samples may be collected at least twotimes between doses and/or at least one time after the final doseadministered to the patient. The cancer cells in each biological sampleobtained are then interrogated for relative percentage of CD5⁺ cancercells to determine whether the amount and/or the frequency ofadministration of the anti-CD200 antibody are sufficient to maintain areduced concentration of CD5⁺ cancer cells. Armed with information onCD5⁺ cancer cell concentration in the patient over time and the effecton the concentration of CD5⁺ cancer cells over time by administering theanti-CD200 antibody to the patient, a medical practitioner (and/or acomputer) can determine an anti-CD200 antibody dosing schedule for thepatient that is sufficient to maintain a reduced concentration of CD5⁺cancer cells in the patient over the course of the treatment. Asdescribed above, the treatment can be performed in conjunction with ananti-CD20 therapy such as rituximab.

An antibody described herein (e.g., an anti-CD20 antibody or ananti-CD200 antibody) can be administered as a fixed dose, or in amilligram per kilogram (mg/kg) dose. In some embodiments, the dose canalso be chosen to reduce or avoid production of antibodies or other hostimmune responses against one or more of the active antibodies in thecomposition. While in no way intended to be limiting, exemplary dosagesof an antibody include, e.g., 1-100 μg/kg, 0.5-50 μg/kg, 0.1-100 μg/kg,0.5-25 μg/kg, 1-20 μg/kg, and 1-10 μg/kg, 1-100 mg/kg, 0.5-50 mg/kg,0.1-100 mg/kg, 0.5-25 mg/kg, 1-20 mg/kg, and 1-10 mg/kg. Exemplarydosages of an antibody described herein include, without limitation, 0.1μg/kg, 0.5 μg/kg, 1.0 μg/kg, 2.0 μg/kg, 4 μg/kg, and 8 μg/kg, 0.1 mg/kg,0.5 mg/kg, 1.0 mg/kg, 2.0 mg/kg, 4 mg/kg, and 8 mg/kg. Exemplary dosesalso include, e.g., greater than or equal to 50 mg/m², 75 mg/m², 100mg/m², 150 mg/m², 200 mg/m², 250 mg/m², 300 mg/m², 350 mg/m², 400 mg/m²,450 mg/m², 500 mg/m², 550 mg/m², and/or 600 mg/m².

A pharmaceutical composition can include a therapeutically effectiveamount of an antibody described herein. Such effective amounts can bereadily determined by one of ordinary skill in the art based, in part,on the effect of the administered antibody, or the combinatorial effectof the antibody and one or more additional active agents, if more thanone agent is used. A therapeutically effective amount of an antibodydescribed herein can also vary according to factors such as the diseasestate, age, sex, and weight of the individual, and the ability of theantibody (and one or more additional active agents) to elicit a desiredresponse in the individual, e.g., amelioration of at least one conditionparameter, e.g., amelioration of at least one symptom of the cancerand/or the presence of at least one of the immunomodulatory effectbiomarkers described herein. A therapeutically effective amount is alsoone in which any toxic or detrimental effects of the composition areoutweighed by the therapeutically beneficial effects.

Toxicity and therapeutic efficacy of such compositions can be determinedby known pharmaceutical procedures in cell cultures or experimentalanimals (e.g., animal models of cancer or autoimmune disorders). Theseprocedures can be used, e.g., for determining the LD₅₀ (the dose lethalto 50% of the population) and the ED₅₀ (the dose therapeuticallyeffective in 50% of the population). The dose ratio between toxic andtherapeutic effects is the therapeutic index and it can be expressed asthe ratio LD₅₀/ED₅₀. An anti-CD200 antibody and/or anti-CD20 therapeuticagent (e.g., an anti-CD20 antibody) that exhibits a high therapeuticindex is preferred. While compositions that exhibit toxic side effectsmay be used, care should be taken to design a delivery system thattargets such compounds to the site of affected tissue and to minimizepotential damage to normal cells and, thereby, reduce side effects.

The following examples are intended to illustrate, not limit, theinvention.

EXAMPLES Example 1 Efficacy of an Anti-CD200 Antibody in a Mouse Modelof Autoimmune Hemolytic Disease

Study 0 (Prevention Model).

Therapeutic anti-CD200 antibodies were tested for their ability toprevent, delay, or lessen the severity of, the production ofautoantibodies associated with autoimmune hemolytic disease using amouse model of the disease. See, e.g., Playfair and Marshall-Clarke(1973) Nat New Biol 243:213-214; Naysmith et al. (1981) Immunol Rev55:55-87.

To elicit in mice the production of autoantibodies that bind to mousered blood cells (RBCs), 2×10⁸ rat RBCs were administeredintraperitoneally (i.p.) to female C57BL/6 mice once on study day 0 andthen once per week thereafter for the remainder of the study. Productionof anti-rat RBC alloantibodies by the immunized mice was observed by thesecond week of the study and production by the mice of anti-mouse RBCautoantibodies was observed by week three.

The rat RBC-immunized mice were divided into six experimental groupsdesignated: Group 1 (six mice), Group 2 (6 mice), Group 3 (8 mice),Group 4 (7 mice), Group 5 (9 mice), and Group 6 (9 mice). One additionalgroup—Group 7 (6 mice)—was also evaluated as a control. The Group 7 micewere neither immunized with rat RBCs nor did they receive any of theadditional treatments described below.

Starting at day 0 (that is the day of the first administration of therat RBCs), the mice of each of Groups 2 to 6 were administered atherapeutic agent or vehicle under the following schedule: for each weekof the study, five doses of agent or vehicle administered as one doseper day for five consecutive days. Group 1 mice were treated with onlyvehicle—phosphate-buffered saline (PBS). Group 2 mice were treated underthe above treatment schedule using 5 mg/kg of a Control antibody thatdoes not bind to CD200, but possesses effector function (IgG2a). Group 3mice were treated under the aforementioned treatment schedule withAntibody 1—an anti-CD200 antibody (IgG2a) having effector function—eachdose being 5 mg/kg. Group 4 mice were treated with cyclosporine at adose of 15 mg/kg. Group 5 mice were treated with the Control Antibody at5 mg/kg and cyclosporine at 15 mg/kg. Group 6 mice were treated withAntibody 1 at a dose of 5 mg/kg and cyclosporine at a dose of 15 mg/kg.The antibody treatments were administered i.p. Cyclosporine wasadministered to the mice subcutaneously (s.c.). The Group design andtreatment schedules for each group are summarized in Table 1.

TABLE 1 Group Design and Treatment Schedule for Study 0. Groups NTherapeutic Administered Dose Group 1 6 Vehicle N/A Group 2 6 Controlantibody (IgG2a) that does 5 mg/kg not bind to CD200 but possesseseffector function Group 3 8 Antibody 1 (anti-CD200 antibody 5 mg/kgIgG2a with effector function) Group 4 7 Cyclosporine 15 mg/kg  Group 5 9Control antibody (IgG2a) that does 5 mg/kg not bind to CD200 butpossesses effector function; and Cyclosporine 15 mg/kg  Group 6 9Antibody 1 (anti-CD200 antibody 5 mg/kg IgG2a with effector function);and Cyclosporine 15 mg/kg  Group 7 6 Non-immunized, non-treated N/Acontrol group N refers to the number of mice in each group. N/A = notapplicable.

On a weekly basis, blood was drawn from the mice of Groups 1 to 7 priorto, during, and after the above treatments to evaluate by flow cytometrywhether treatment affected the titer of anti-mouse RBC autoantibodiesand/or anti-rat RBC alloantibodies in the mice. To determine therelative concentration of anti-mouse autoantibodies produced in asubject mouse (e.g., a treated mouse from Group 3), whole blood obtainedfrom the mouse was incubated with a preparation of fluorescently-labeledanti-mouse antibody to thereby detect the presence of anti-mouse RBCantibodies present on the surface of mouse RBC in the blood of theanimals. The cells were washed with PBS and then subjected to flowcytometry to evaluate the relative amount of mouse anti-mouse RBCs boundto the mouse RBCs as a function of the mean fluorescence intensity.Between day 13 and 27, the concentration of anti-mouse RBCautoantibodies in the mice of Groups 1, 2, 4, 5, and 6 increased. Incontrast, the concentration of anti-mouse RBC autoantibodies in the miceof Group 3 was markedly reduced as compared to the concentration ofautoantibody in the other groups. In addition, the production ofautoantibody by the mice in Group 3 was markedly delayed as compared tothe mice in the other groups (FIG. 1). For example, 50% of mice inGroups 1, 2, 4, 5, and 6 developed autoantibodies between day 20 and 27of the study. In contrast, autoantibody production in at least 50% ofmice in Group 3 did not occur until between day 27 and day 34. Theseresults indicate that Antibody 1, an anti-CD200 antibody, at 5 mg/kg wascapable of not only reducing the concentration of anti-mouse RBCautoantibodies in a mice model of autoimmune hemolytic disease, but wasalso capable of delaying significantly the production of theautoantibodies in the mice.

To determine the relative concentration of alloantibodies produced in asubject mouse (e.g., a treated mouse from Group 3), serum obtained fromthe mouse was incubated with a sample of isolated rat RBCs for a timeand under conditions sufficient for any rat RBC-specific alloantibodiespresent in the serum to bind to the rat RBCs. The cells were washed withPBS and then incubated with a fluorescently-labeled antibody that bindsto mouse antibodies. Following an additional washing step, the cellswere subjected to flow cytometry to evaluate the relative amount ofmouse anti-rat RBCs bound to the rat RBCs as the mean fluorescenceintensity. Sera obtained from mice of Groups 1, 2, 4, 5, and 6 containedan increasing concentration of anti-rat RBC alloantibodies over thecourse of the experiment. In contrast, sera obtained from the mice ofGroup 3 contained much less detectable anti-rat RBC autoantibodies ascompared to the other Groups. These results further indicated thatAntibody 1, an anti-CD200 antibody, at 5 mg/kg was capable of reducingthe titer of RBC-specific alloantibodies, as well as anti-RBCautoantibodies, produced in a mouse model of autoimmune hemolyticdisease.

Study 1 (Treatment Model).

Therapeutic anti-CD200 antibodies were tested for their ability toreduce the production of autoantibodies associated with autoimmunehemolytic disease using a mouse model of the disease. To elicit in micethe production of autoantibodies that bind to mouse red blood cells(RBCs), 2×10⁸ rat RBCs were administered intraperitoneally (i.p.) tofemale C57BL/6 mice once on study day 0 and then once per weekthereafter for the remainder of the study. Production of anti-rat RBCalloantibodies by the immunized mice was observed by the second week ofthe study and production by the mice of anti-mouse RBC autoantibodieswas observed by week three.

The rat RBC-immunized mice were divided into five groups designatedGroup 1 (8 mice), Group 2 (8 mice), Group 3 (8 mice), Group 4 (7 mice),and Group 5 (8 mice). A sixth group of mice (designated Group 6; 6 mice)was also evaluated as a control. The Group 6 mice were neither immunizedwith rat RBCs nor did they receive any of the additional treatmentsdescribed below.

Starting on day 112, the mice of each of Groups 1 to 5 received anadditional treatment of 14 doses of a therapeutic agent or vehiclecontrol administered under the following schedule: (i) five doses ofagent or vehicle administered as one dose per day for five consecutivedays; (ii) a two day break in treatment; (iii) an additional five dosesof the agent or vehicle administered one dose per day for fiveconsecutive days; another two day break in treatment; and (iv) four moredoses of agent or vehicle administered one dose per day for fourconsecutive days. Group 1 mice were treated with onlyvehicle—phosphate-buffered saline (PBS). Group 2 mice were treated underthe aforementioned treatment schedule with Antibody 1—an anti-CD200antibody (IgG2a) having effector function—each dose being 5 mg/kg. Group3 mice were treated with Antibody 1 at a dose of 1 mg/kg. Group 4 micewere treated under the above treatment schedule with Antibody 2—ananti-CD200 antibody that lacked effector function—each dose at 5 mg/kg.Group 5 mice were treated under the above treatment schedule using adose of 5 mg/kg of a Control antibody that does not bind to CD200, butpossesses effector function (IgG2a). The Group design and treatmentschedules for each group are summarized in Table 2.

TABLE 2 Group Design and Treatment Schedule for Study 1. Groups NTherapeutic Administered Dose Group 1 8 Vehicle N/A Group 2 8 Antibody 1(anti-CD200 antibody 5 mg/kg IgG2a with effector function) Group 3 8Antibody 1 (anti-CD200 antibody 1 mg/kg IgG2a with effector function)Group 4 7 Antibody 2 (anti-CD200 antibody 5 mg/kg that does not possesseffector function) Group 5 8 Control antibody (IgG2a) that does 5 mg/kgnot bind to CD200 but possesses effector function Group 6 6Non-immunized, non-treated N/A control group N refers to the number ofmice in each group. N/A = not applicable.

On a weekly basis, blood was drawn from the mice of Groups 1 to 6 priorto, during, and after the above treatments to evaluate by flow cytometrywhether treatment affected the titer of anti-mouse RBC autoantibodiesand/or anti-rat RBC alloantibodies in the mice. Between day 133 and 137of the study, the mice were sacrificed and their spleens harvested. Todetermine the relative concentration of alloantibodies produced in asubject mouse (e.g., a treated mouse from Group 2), serum obtained fromthe mouse (e.g., at day 133) was contacted to a sample of isolated ratRBCs for a time and under conditions sufficient for any rat RBC-specificalloantibodies present in the serum to bind to the rat RBCs. The cellswere washed with PBS and then incubated with a fluorescently-labeledantibody that binds to mouse antibodies. Following an additional washingstep, the cells were subjected to flow cytometry to evaluate therelative amount of mouse anti-rat RBCs bound to the rat RBCs as the meanfluorescence intensity. The inventors observed that the post-treatmentsera obtained from mice of Groups 1, 3, 4, and 5 contained an increasedconcentration of anti-rat RBC alloantibodies as compared to thecorresponding sera obtained from the mice prior to treatment. Incontrast, sera obtained from the mice of Group 2 post-treatmentcontained less detectable anti-rat RBC alloantibodies as compared to thecorresponding sera obtained from the mice prior to treatment. Theseresults indicated that Antibody 1, an anti-CD200 antibody, at 5 mg/kgwas capable of reducing the production of RBC-specific antibodies in amouse model of autoimmune hemolytic disease.

The inventors subsequently observed that Antibody 2 had a significantlyshorter half-life in the treated mice as compared to the half-life ofAntibody 1. Thus the results observed with Antibody 2 in Study 1 and inother studies described herein may not fully reflect the true efficacyof the Antibody 2 in the autoimmune hemolytic disease model nor theimmunodulatory effect of the antibody in animals.

Study 2 (Prevention Model).

Therapeutic anti-CD200 antibodies were tested for their ability toprevent, delay, or lessen the severity of, the production ofautoantibodies associated with autoimmune hemolytic disease using theabove-described mouse model of the disease.

To elicit in mice the production of autoantibodies that bind to mousered blood cells (RBCs), rat RBCs were administered intraperitoneally(i.p.) to female BALB/c mice once on study day 0 and then once per weekthereafter for the remainder of the study. As described above,production of anti-rat RBC alloantibodies by the immunized mice wasobserved by the second week of the study and production by the mice ofanti-mouse RBC autoantibodies was observed by week three.

The rat RBC-immunized mice were divided into five groups designatedGroup 1 (8 mice), Group 2 (8 mice), Group 3 (8 mice), Group 4 (8 mice),and Group 5 (8 mice). A sixth group of mice (designated Group 6; 6 mice)was also evaluated as a control. The Group 6 mice were neither immunizedwith rat RBCs nor did they receive any of the additional treatmentsdescribed below.

Starting at day 0 (that is the day of the first administration of therat RBCs), the mice of each of Groups 1 to 5 were administered atherapeutic agent or vehicle under the following schedule: for each weekof the study, five doses of agent or vehicle administered as one doseper day for five consecutive days. Group 1 mice were treated with onlyvehicle—phosphate-buffered saline (PBS). Group 2 mice were treated underthe aforementioned treatment schedule with Antibody 1—an anti-CD200antibody (IgG2a) having effector function—each dose being 5 mg/kg. Group3 mice were treated with Antibody 1 at a dose of 1 mg/kg. Group 4 micewere treated under the above treatment schedule with Antibody 2—ananti-CD200 antibody that lacked effector function—each dose at 5 mg/kg.Group 5 mice were treated under the above treatment schedule using 5mg/kg of a Control antibody that does not bind to CD200, but possesseseffector function (IgG2a). The Group design and treatment schedules foreach group are summarized in Table 3.

TABLE 3 Group Design and Treatment Schedule for Study 2. Groups NTherapeutic Administered Dose Group 1 8 Vehicle N/A Group 2 8 Antibody 1(anti-CD200 antibody 5 mg/kg IgG2a with effector function) Group 3 8Antibody 1 (anti-CD200 antibody 1 mg/kg IgG2a with effector function)Group 4 8 Antibody 2 (anti-CD200 antibody 5 mg/kg that does not possesseffector function) Group 5 8 Control antibody (IgG2a) that does 5 mg/kgnot bind to CD200 but possesses effector function Group 6 6Non-immunized, non-treated N/A control group N refers to the number ofmice in each group. N/A = not applicable.

On a weekly basis, blood was drawn from the mice of Groups 1 to 6 priorto, during, and after the above treatments to evaluate by flow cytometrywhether treatment affected the titer of anti-mouse RBC autoantibodiesand/or anti-rat RBC alloantibodies in the mice. On day 64 or 65 of thestudy, the mice were sacrificed and their spleens harvested. (Four micein each group were sacrificed on day 64 and the other four mice in eachgroup were sacrificed on day 65.) To determine the relativeconcentration of alloantibodies produced in a subject mouse (e.g., atreated mouse from Group 3), serum obtained from the mouse was contactedto a sample of isolated rat RBCs for a time and under conditionssufficient for any rat RBC-specific alloantibodies present in the serumto bind to the rat RBCs. The cells were washed with PBS and thenincubated with a fluorescently-labeled antibody that binds to mouseantibodies. Following an additional washing step, the cells weresubjected to flow cytometry to evaluate the relative amount of mouseanti-rat RBCs bound to the rat RBCs as the mean fluorescence intensity.As shown in FIG. 2, sera obtained from mice of Groups 1, 3, 4, and 5contained an increasing concentration of anti-rat RBC alloantibodiesover the course of the experiment. In contrast, sera obtained from themice of Group 2 post-treatment contained much less detectable anti-ratRBC alloantibodies as compared to the other Groups. These resultsfurther indicated that Antibody 1, an anti-CD200 antibody, at 5 mg/kgwas capable of reducing the titer of RBC-specific alloantibodiesproduced in a mouse model of autoimmune hemolytic disease.

Study 3 (Treatment Model).

Therapeutic anti-CD200 antibodies were tested for their ability to treatautoimmune hemolytic disease using a mouse model of the disease. Toelicit in mice the production of autoantibodies that bind to mouse redblood cells (RBCs), rat RBCs were administered intraperitoneally (i.p.)to female C57BL/6 mice once on study day 0 and then once per weekthereafter for the remainder of the study. As described above,production of anti-rat RBC alloantibodies by the immunized mice wasobserved by the second week of the study and production by the mice ofanti-mouse RBC autoantibodies was observed by week three. The ratRBC-immunized mice were divided into three groups designated Group 1 (6mice), Group 2 (3 mice), and Group 3 (5 mice).

Starting on day 86, the mice of each of Groups 1 to 3 received anadditional treatment of 10 doses of a therapeutic agent or vehiclecontrol administered under the following schedule: (i) five doses ofagent or vehicle administered as one dose per day for five consecutivedays; (ii) a two day break in treatment; and (iii) an additional fivedoses of the agent or vehicle administered one dose per day for fiveconsecutive days. Group 1 mice were treated under the aforementionedtreatment schedule with Antibody 1—an anti-CD200 antibody (IgG2a) havingeffector function—each dose being 5 mg/kg. Group 2 mice were treatedwith Antibody 1 at a dose of 1 mg/kg. Group 3 mice were treated underthe above treatment schedule with Antibody 2—an anti-CD200 antibody thatlacked effector function—each dose at 5 mg/kg. The Group design andtreatment schedules for each group are summarized in Table 4.

TABLE 4 Group Design and Treatment Schedule for Study 3. Groups NTherapeutic Administered Dose Group 1 6 Antibody 1 (anti-CD200 antibody5 mg/kg IgG2a with effector function) Group 2 3 Antibody 1 (anti-CD200antibody 1 mg/kg IgG2a with effector function) Group 3 5 ControlAntibody 5 mg/kg Group 4 3 Non-immunized, non-treated N/A control groupN refers to the number of mice in each group. N/A = not applicable.

At the conclusion of the study, the mice were sacrificed and theirspleens harvested. To determine whether administration of Antibody 1 tothe mice affected activation of splenocytes by RBC, in addition toaffecting the production of anti-RBC antibodies in the mice, splenocyteactivation in the presence of RBCs was evaluated using an in vitroproliferation assay. Briefly, isolated splenocytes were cultured withone of three different antigens—mouse RBCs, rat RBCs, or bovine serumalbumin (control)—or with media alone. Following contact of thesplenocytes with the antigens, ³H-thymidine was added to the splenocyteculture media for approximately 16 hours. The media was removed and thecells harvested. The relative activation of the splenocytes by theantigens was then measured as a function of the amount of ³H-thymidineincorporated into the DNA of the splenocytes.

As shown in FIG. 3, splenocytes from Group 2 and 3 mice exhibited arobust proliferative response following contact with rat RBCs. Incontrast, splenocytes from Group 1 mice proliferated very little in thepresence of rat RBCs indicating that administration of an anti-CD200antibody at 5 mg/kg was capable of inhibiting the activation ofsplenocytes by rat RBCs in a mouse model of autoimmune hemolyticdisease.

Study 4 (Treatment Model).

As described above, to elicit in mice the production of autoantibodiesthat bind to mouse red blood cells (RBCs), rat RBCs were administeredintraperitoneally (i.p.) to female C57BL/6 mice once on study day 0 andthen once per week thereafter for the remainder of the study.

The rat RBC-immunized mice were divided into seven (7) groups designatedGroup 1, Group 2, Group 3, Group 4, Group 5, Group 6, and Group 7. Aneighth group of mice (designated Group 8) was also evaluated as acontrol. The Group 8 mice were neither immunized with rat RBCs nor didthey receive any of the additional treatments described below. Ten micewere in each group.

Starting on day 21, the mice of each of Groups 1 to 7 received anadditional treatment of one or more therapeutic agents or vehiclecontrol administered under the following schedule: for each week of thestudy, five doses of one or more agents or vehicle administered as onedose per day for five consecutive days. Group 1 mice were treated withonly vehicle—phosphate-buffered saline (PBS). Group 2 mice were treatedunder the above treatment schedule using a dose of 5 mg/kg of a Controlantibody that does not bind to CD200, but possesses effector function(IgG2a). Group 3 mice were treated under the aforementioned treatmentschedule with Antibody 1—an anti-CD200 antibody (IgG2a) having effectorfunction—each dose being 5 mg/kg. Group 4 mice were treated under theabove schedule with 15 mg/kg cyclosporine. Group 5 mice were treatedunder the above dosing schedule with both the Control antibody (at 5mg/kg) and cyclosporine (at 15 mg/kg). Group 6 mice were treated underthe above dosing schedule with both Antibody 1 (at 5 mg/kg) andcyclosporine (at 15 mg/kg). Group 7 mice were treated under the abovedosing schedule with both Antibody 1 (at 1 mg/kg) and cyclosporine (at15 mg/kg). The Group design and treatment schedules for each group aresummarized in Table 5.

TABLE 5 Group Design and Treatment Schedule for Study 4. Groups NTherapeutic Administered Dose Group 1 10 Vehicle N/A Group 2 10 Controlantibody (IgG2a) that does 5 mg/kg not bind to CD200 but possesseseffector function Group 3 10 Antibody 1 (anti-CD200 antibody 5 mg/kgIgG2a with effector function) Group 4 10 Cyclosporine 15 mg/kg  Group 510 Control antibody; and 5 mg/kg cyclosporine 15 mg/kg  Group 6 10Antibody 1; and 5 mg/kg cyclosporine 15 mg/kg  Group 7 10 Antibody 1;and 1 mg/kg cyclosporine 15 mg/kg  Group 8 10 Non-immunized, non-treatedN/A control group N refers to the number of mice in each group. N/A =not applicable.

On a weekly basis, blood was drawn from the mice of Groups 1 to 8 priorto, during, and after the above treatments to evaluate by flow cytometrywhether treatment affected the titer of anti-mouse RBC autoantibodiesand/or anti-rat RBC alloantibodies in the mice. On day 37 of the study,the mice were sacrificed and their spleens harvested. Bone marrow cellswere also obtained from the two mouse femur and tibia bones. The spleenand bone marrow cells were subjected to flow cytometry as describedbelow (Example 2).

A reduced concentration of anti-rat RBC alloantibodies was present inpost-treatment sera obtained from mice of Groups 3 and 4 as compared tothe corresponding pre-treatment sera. The post-treatment reduction inanti-rat RBC alloantibodies was even greater in the mice of Groups 6 and7, indicating that cyclosporine and Antibody 1 have a synergistic effecton reducing alloantibody production in the mice. These results evenfurther indicated that an anti-CD200 antibody was capable of reducingthe titer of RBC-specific antibodies produced in a mouse model ofautoimmune hemolytic disease and that an anti-CD200 antibody is usefulfor treating the disease.

Example 2 Administration of an Anti-CD200 Antibody to Mice Affects theConcentration of Splenocyte and Bone Marrow Cell Populations in the Mice

Splenocytes obtained from the mice of Study 1 were evaluated todetermine the percentage of cells that express CD200. Cells wereharvested from the spleens of the mice and incubated with a compositionof biotin-labeled anti-CD200 antibodies (polyclonal) for an amount oftime and under conditions sufficient to allow for binding of theantibodies to CD200, if present on the cells. The polyclonal antibodypreparation was used to prevent or lessen any masking effect due to thepresence of residual therapeutic anti-CD200 antibody (e.g., Antibody 1or Antibody 2) on the cells. The cells were washed and incubated with afluorescently-labeled streptavidin moiety. Following an additionalwashing step, the cells were then subjected to flow cytometry. As shownin FIG. 4, there was a marked reduction in the concentration of CD200⁺splenocytes in mice treated with 14, 5 mg/kg doses of Antibody 1 ascompared to the concentration of CD200⁺ splenocytes in mice treated withvehicle, the Control antibody, or Antibody 2.

Splenocytes harvested from the spleens of the mice of Study 2 were alsosubjected to staining and flow cytometry analysis as described above.There was a marked reduction in the concentration of CD200⁺ splenocytesin mice chronically treated with 5 mg/kg of Antibody 1, as compared tothe concentration of CD200⁺ splenocytes in mice treated with vehicle orthe Control antibody. There was also no change in the concentration ofCD200⁺ splenocytes in the Group 3 mice treated with 1 mg/kg dose ofAntibody 1 and Group 4 mice trated with 5 mg/kg Antibody 2.

Splenocytes harvested from the spleens of the mice of Study 4 were alsosubjected to staining and flow cytometry analysis as described above.There was a marked reduction in the concentration of CD200⁺ splenocytesin mice treated with 5 mg/kg of Antibody 1 with or without cyclosporine,as compared to the concentration of CD200⁺ splenocytes in mice treatedwith vehicle, the Control antibody, cyclosporine alone, or a combinationof the Control antibody and cyclosporine. There was also no change inthe concentration of CD200⁺ splenocytes in the Group 7 mice treated witha combination schedule of cyclosporine and a 1 mg/kg dose of Antibody 1.An analysis of the mean fluorescence intensity (MFI) of the CD200⁺splenocytes from each mouse (which is a measure of the relativeexpression level of CD200 by each CD200⁺ splenocyte) was also performed.The MFI of CD200⁺ splenocytes from Groups 4 and 7 was markedly reducedas compared to the MFI of CD200⁺ splenocytes in the remaining Groups(save Group 8). This indicated that not only does administration ofAntibody 1 to the mice reduce the total number of CD200⁺ splenocytes,but the remaining cells that do express CD200⁺ in Antibody 1-treatedmice express CD200 at much lower levels.

Taken together, these results confirm that administration of ananti-CD200 antibody to an animal reduces the concentration of CD200⁺splenocytes in the animal. The results also indicate that the anti-CD200antibody-mediated reduction in CD200⁺ splenocytes is not positively ornegatively affected by cyclosporine.

The inventors also further examined the effect of anti-CD200 antibodieson: (i) the concentration of particular CD200⁺ lymphocyte subsets ofsplenocytes from the mice of Study 4 and (ii) the concentration ofparticular CD200⁺ subsets of bone marrow-derived cells from the mice ofStudy 4.

Concentration of Splenic Lymphocyte Subsets in the Mice of Study 4

CD3⁺/CD200⁺ Lymphocyte Subset.

A sample of splenocytes from each of the mice of Study 4 was incubatedwith the polyclonal anti-CD200 antibody preparation and adetectably-labeled antibody that binds to CD3 to thereby identify theproportion of CD3⁺/CD200⁺ cells in the spleens of mice from Groups 1 to8. The CD3⁺ population of cells includes T cells such as CD4⁺ and CD8⁺ Tcells. The labeled cells were subjected to flow cytometry. There was amarked reduction in the concentration of CD3⁺/CD200⁺ splenocytes in micechronically treated with 5 mg/kg of Antibody 1 with or withoutcyclosporine, as compared to the concentration of CD3⁺/CD200⁺splenocytes in mice treated with vehicle, the Control antibody,cyclosporine alone, or a combination of the Control antibody andcyclosporine. There was also no significant change in the concentrationof CD3⁺/CD200⁺ splenocytes in the Group 7 mice treated with acombination schedule of cyclosporine and a 1 mg/kg dose of Antibody 1.

CD5⁺/CD200⁺ Lymphocyte Subset.

A sample of splenocytes from each of the mice of Study 4 was incubatedwith the polyclonal anti-CD200 antibody preparation and adetectably-labeled antibody that binds to CD5 to thereby identify theproportion of CD5⁺/CD200⁺ cells in the spleens of mice from Groups 1 to8. The CD5⁺ population of cells includes T cells as well as B cells (theB1 cell population). The labeled cells were subjected to flow cytometry.There was a marked reduction in the concentration of CD5⁺/CD200⁺splenocytes in mice chronically treated with 5 mg/kg of Antibody 1 withor without cyclosporine, as compared to the concentration of CD5⁺/CD200⁺splenocytes in mice treated with vehicle, the Control antibody,cyclosporine alone, or a combination of the Control antibody andcyclosporine. There was also no significant change in the concentrationof CD5⁺/CD200⁺ splenocytes in the Group 7 mice treated with acombination schedule of cyclosporine and a 1 mg/kg dose of Antibody 1.

CD19⁺/CD200⁺ Lymphocyte Subset.

A sample of splenocytes from each of the mice of Study 4 was incubatedwith the polyclonal anti-CD200 antibody preparation and adetectably-labeled antibody that binds to CD19 to thereby identify theproportion of CD19⁺/CD200⁺ cells in the spleens of mice from Groups 1 to8. The CD19⁺ population of cells includes B cells. The labeled cellswere subjected to flow cytometry. Like CD5⁺/CD200⁺ cells and CD3⁺/CD200⁺cells, there was also a marked reduction in the concentration ofCD19⁺/CD200⁺ splenocytes in mice chronically treated with 5 mg/kg ofAntibody 1 with or without cyclosporine, as compared to theconcentration of CD19⁺/CD200⁺ splenocytes in mice treated with vehicle,the Control antibody, cyclosporine alone, or a combination of theControl antibody and cyclosporine. There was also no significant changein the concentration of CD19⁺/CD200⁺ splenocytes in the Group 7 micetreated with a combination schedule of cyclosporine and a 1 mg/kg doseof Antibody 1.

CD138⁺/CD200⁺ Lymphocyte Subset.

A sample of splenocytes from each of the mice of Study 4 was incubatedwith the polyclonal anti-CD200 antibody preparation and adetectably-labeled antibody that binds to CD138 to thereby identify theproportion of CD138⁺/CD200⁺ cells in the spleens of mice from Groups 1to 8. The CD138⁺ population of cells includes plasma cells. The labeledcells were subjected to flow cytometry. There was a marked reduction inthe concentration of CD138⁺/CD200⁺ splenocytes in mice chronicallytreated with 5 mg/kg of Antibody 1 with or without cyclosporine, ascompared to the concentration of CD138⁺/CD200⁺ splenocytes in micetreated with vehicle, the Control antibody, cyclosporine alone, or acombination of the Control antibody and cyclosporine. There was also nosignificant change in the concentration of CD138⁺/CD200⁺ splenocytes inthe Group 7 mice treated with a combination schedule of cyclosporine anda 1 mg/kg dose of Antibody 1.

F4/80⁺ Lymphocyte Subset.

F4/80 is 125 kDa transmembrane protein present on the cell-surface ofmature mouse macrophages. To determine whether administration of ananti-CD200 antibody affects the concentration of resident macrophages inspleen, a sample of splenocytes from each mouse of Study 4 was incubatedwith a detectably-labeled antibody that binds to F4/80. The labeledcells were subjected to flow cytometry to thereby identify theproportion of F4/80⁺ cells in the spleens of mice from Groups 1 to 8.The concentration of F4/80⁺ splenocytes increased in mice treated with 5mg/kg of Antibody 1 (10 doses) with or without cyclosporine, as comparedto the concentration of F4/80⁺ splenocytes in mice treated with vehicle,the Control antibody, cyclosporine alone, or a combination of theControl antibody and cyclosporine. There was also no significant changein the concentration of F4/80⁺ splenocytes in the Group 7 mice treatedwith a combination schedule of cyclosporine and a 1 mg/kg dose ofAntibody 1.

Taken together, these results indicate that administration of ananti-CD200 antibody reduces a variety of CD200⁺ splenocyte subsets, butincreases certain macrophage subsets, in the treated animals.

Concentration of Bone Marrow Cell Subsets in the Mice of Study 4

CD34⁺/CD200⁺ Bone Marrow Cell Subset.

A sample of bone marrow cells from each of the mice was incubated withthe polyclonal anti-CD200 antibody preparation and a detectably-labeledantibody that binds to CD34 to thereby identify the proportion ofCD34⁺/CD200⁺ cells in the bone marrow of mice from Groups 1 to 8. TheCD34⁺ cells include a population of hematopoietic stem cells (HSCs). Thelabeled cells were subjected to flow cytometry also selecting for thosecells that are lineage low (Lin^(−/Low)). There was a marked reductionin the concentration of CD34⁺/CD200⁺ bone marrow cells in mice treatedwith 5 mg/kg of Antibody 1 (10 doses) with or without cyclosporine, ascompared to the concentration of CD34⁺/CD200⁺ bone marrow cells in micetreated with vehicle, the Control antibody, cyclosporine alone, or acombination of the Control antibody and cyclosporine. There was also nosignificant change in the concentration of CD34⁺/CD200⁺ bone marrowcells in the Group 7 mice treated with a combination schedule ofcyclosporine and a 1 mg/kg dose of Antibody 1.

Sca-1⁺/CD200⁺ Bone Marrow Cell Subsets.

A sample of bone marrow cells from each of the mice was incubated withthe polyclonal anti-CD200 antibody preparation and a detectably-labeledantibody that binds to Sca-1 to thereby identify the proportion ofSca-1⁺/CD200⁺ cells in the bone marrow of mice from Groups 1 to 8. TheSca-1⁺ cells include a population of HSCs and mesenchymal stem cells(MSCs). The labeled cells were subjected to flow cytometry alsoselecting for those cells that are lineage low (Lin^(−/Low)). There wasa marked reduction in the concentration of Sca-1⁺/CD200⁺ bone marrowcells in mice treated with 5 mg/kg of Antibody 1 (10 doses) with orwithout cyclosporine, as compared to the concentration of Sca-1⁺/CD200⁺bone marrow cells in mice treated with vehicle, the Control antibody,cyclosporine alone, or a combination of the Control antibody andcyclosporine. There was also no significant change in the concentrationof Sca-1⁺/CD200⁺ bone marrow cells in the Group 7 mice treated with acombination schedule of cyclosporine and a 1 mg/kg dose of Antibody 1.

Sca-1⁺/CD34⁺ Bone Marrow Cell Subsets.

A sample of bone marrow cells from each of the mice was incubated with afirst detectably-labeled antibody that binds to CD34 and a seconddetectably-labeled antibody that binds to Sca-1 to thereby identify theproportion of Sca-1⁺/CD34⁺ cells in the bone marrow of mice from Groups1 to 8. The labeled cells were subjected to flow cytometry alsoselecting for those cells that are lineage low (Lin^(−/Low)). TheSca-1⁺/CD34⁺/Lin− cells include a population of MSCs. There was a markedreduction in the concentration of Sca-1⁺/CD34⁺ bone marrow cells in micetreated with 5 mg/kg of Antibody 1 (10 doses) with or withoutcyclosporine, as compared to the concentration of Sca-1⁺/CD34⁺ bonemarrow cells in mice treated with vehicle, the Control antibody,cyclosporine alone, or a combination of the Control antibody andcyclosporine. There was also no significant change in the concentrationof Sca-1⁺/CD34⁺ bone marrow cells in the Group 7 mice treated with acombination schedule of cyclosporine and a 1 mg/kg dose of Antibody 1.

c-kit⁺/CD200⁺ Bone Marrow Cell Subsets.

A sample of bone marrow cells from each of the mice was incubated withthe polyclonal anti-CD200 antibody preparation and a detectably-labeledantibody that binds to c-kit to thereby identify the proportion ofc-kit⁺/CD200⁺ cells in the bone marrow of mice from Groups 1 to 8. Thec-kit⁺ cells include a population of HSCs and MSCs. The labeled cellswere subjected to flow cytometry also selecting for those cells that arelineage low (Lin^(−/Low)). There was a marked reduction in theconcentration of c-kit⁺/CD200⁺ bone marrow cells in mice chronicallytreated with 5 mg/kg of Antibody 1 with or without cyclosporine, ascompared to the concentration of c-kit⁺/CD200⁺ bone marrow cells in micetreated with vehicle, the Control antibody, cyclosporine alone, or acombination of the Control antibody and cyclosporine. There was also nosignificant change in the concentration of c-kit⁺/CD200⁺ bone marrowcells in the Group 7 mice treated with a combination schedule ofcyclosporine and a 1 mg/kg dose of Antibody 1.

CD200⁺/CD200R⁺ Bone Marrow Cell Subset.

A sample of bone marrow cells from each of the mice was incubated withthe polyclonal anti-CD200 antibody preparation and a detectably-labeledantibody that binds to CD200R to thereby identify the proportion ofCD200⁺/CD200R⁺ cells in the bone marrow of mice from Groups 1 to 8. Thelabeled cells were subjected to flow cytometry. There was a markedreduction in the concentration of CD200⁺/CD200R⁺ bone marrow cells inmice chronically treated with 5 mg/kg of Antibody 1 with or withoutcyclosporine, as compared to the concentration of CD200⁺/CD200R⁺ bonemarrow cells in mice treated with vehicle, the Control antibody,cyclosporine alone, or a combination of the Control antibody andcyclosporine. There was also no significant change in the concentrationof CD200⁺/CD200R⁺ bone marrow cells in the Group 7 mice treated with acombination schedule of cyclosporine and a 1 mg/kg dose of Antibody 1.

Example 3 Recovery of Bone Marrow Cell and CD200⁺ Splenocyte Subsetsafter Withdrawal of Anti-CD200 Therapy

Study 5 (Treatment Model).

The therapeutic anti-CD200 antibodies were again tested for theirability modulate the concentration of specific subset populations ofsplenocytes and bone marrow cells. The antibodies were administered tothe mice in the context of a mouse model of autoimmune hemolyticdisease. As described above, to elicit in mice the production ofautoantibodies that bind to mouse red blood cells (RBCs), 2×10⁸ rat RBCswere administered intraperitoneally (i.p.) to female BALB/c mice once onstudy day 0 and then once per week thereafter for the remainder of thestudy. Production of anti-rat RBC alloantibodies by the immunized micewas observed by the second week of the study and production by the miceof anti-mouse RBC autoantibodies was observed by week three.

The rat RBC-immunized mice were divided into five groups designatedGroup 2 (20 mice), Group 3 (20 mice), Group 4 (20 mice), Group 5 (15mice), and Group 6 (15 mice). A sixth group of mice (designated Group 1;20 mice) was also evaluated as a control. The Group 1 mice were neitherimmunized with rat RBCs nor did they receive any of the additionaltreatments described below.

Starting on day 21, the mice of each of Groups 2 to 6 received anadditional treatment of 10 doses of a therapeutic agent or vehiclecontrol administered under the following schedule: (i) five doses ofagent or vehicle administered as one dose per day for five consecutivedays; (ii) a two day break in treatment; and (iii) an additional fivedoses of the agent or vehicle administered one dose per day for fiveconsecutive days. Group 6 mice were treated with onlyvehicle—phosphate-buffered saline (PBS). Group 2 mice were treated underthe aforementioned treatment schedule with Antibody 1—an anti-CD200antibody (IgG2a) having effector function—each dose being 5 mg/kg. Group3 mice were treated under the above treatment schedule with Antibody2—an anti-CD200 antibody that lacked effector function—each dose at 5mg/kg. Group 4 mice were treated under the above treatment scheduleusing a dose of 5 mg/kg of a Control antibody that does not bind toCD200, but possesses effector function (IgG2a). Group 5 mice weretreated under the above treatment schedule using a dose of 5 mg/kg of aControl antibody that does not bind to CD200 and does not possesseffector function. The Group design and treatment schedules for eachgroup are summarized in Table 6.

TABLE 6 Group Design and Treatment Schedule for Study 5. Groups NTherapeutic Administered Dose Group 1 20 Non-immunized, non-treated N/Acontrol group Group 2 20 Antibody 1 (anti-CD200 antibody 5 mg/kg IgG2awith effector function) Group 3 20 Antibody 2 (anti-CD200 antibody 5mg/kg that does not possess effector function) Group 4 20 Controlantibody (IgG2a) that does 5 mg/kg not bind to CD200 but possesseseffector function Group 5 15 Control antibody (IgG2a) that does 5 mg/kgnot bind to CD200 and does not possess effector function Group 6 15Vehicle N/A N refers to the number of mice in each group. N/A = notapplicable.

On a weekly basis, blood was drawn from the mice of Groups 1 to 6 priorto, during, and after the above treatments to evaluate by flow cytometrywhether treatment affected the titer of anti-mouse RBC autoantibodiesand/or anti-rat RBC alloantibodies in the mice. On day 35 of the study,three of the mice in each group were sacrificed and their spleensharvested. Bone marrow was also isolated from the femurs and tibias ofeach mouse. As described above, the cells were labeled withdetectably-labeled antibodies (e.g., the polyclonal anti-CD200 antibodypreparation and an additional fluorescently-labeled antibody) andsubjected to flow cytometry. A summary of the results are shown below inTable 7.

TABLE 7 Effect of Anti-CD200 Antibodies on Splenocyte and Bone MarrowCell Subsets at day 35 Cell Subset/ Reduction (R) or Reduction (R) orTissue Expression Increase (I) in Increase (I) in Type Profile Group 2Mice** Group 3 Mice** Spleen CD200⁺ R R Spleen CD3⁺/CD200⁺ R — SpleenCD5⁺/CD200⁺ R — Spleen CD19⁺/CD200⁺ R — Spleen CD45R⁺/CD200⁺ R — SpleenCD138⁺/CD200⁺ R R (Gated on CD45R⁺ cells) Spleen CD200⁺ (Gated on R  R*CD45R⁺ cells) Bone CD200⁺ R — Marrow Bone CDIgk⁺/CD200⁺ R — Marrow BoneCD200⁺ (Gated on R — Marrow CD45R⁺ cells) Bone CD200⁺ (Gated on R —Marrow CD138⁺/CD45R⁻ cells) Bone c-kit⁺/CD200⁺ R R Marrow (Gated on Lin⁻cells) *indicates that the reduction in concentration of a particularcell subset in mice treated with Antibody 2 is not as profound as thereduction observed in the same cell subset in mice treated withAntibody 1. **indicates that the reduction or increase in theconcentration of a particular cell subset is relative the concentrationof the particular subset in vehicle treated mice (Group 6) and thecorresponding isotype control. Thus, the reduction of CD200⁺ splenocytesobserved in mice of Group 2 mice is relative to the concentration ofCD200⁺ splenocytes in Group 6 mice and Group 4 mice. “—” indicates thatno difference in the levels was observed between Antibody 2 and itscorresponding Control antibody.

From day 35 to day 91, the remaining mice in each group receivedadditional RBC immunizations but no treatments with the antibodies, thepurpose being to determine if the populations of splenocytes and bonemarrow cells would recover over time. Three mice in each group weresacrificed at day 91 and their spleens and bone marrow harvested asdescribed above. Flow cytometry analysis was performed on the isolatedcells to determine whether particular population subsets of splenocytesand bone marrow cells, which were reduced at day 35, recovered by day91. Each of the cell populations recovered fully by day 91, indicatingthat the modulatory effects of the anti-CD200 antibody on theconcentration of bone marrow cell and splenocyte subsets is reversibleupon withdrawal of the antibody.

While the present disclosure has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of thedisclosure. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the presentdisclosure. All such modifications are intended to be within the scopeof the disclosure.

What is claimed is:
 1. A method for treating a human afflicted with acancer, the method comprising: (a) identifying a human having a cancerthat is resistant to anti-CD20 antibody therapy, and (b) administeringto the human an anti-CD200 antibody, or a CD200-binding fragmentthereof, in an amount that is sufficient to treat the cancer.
 2. Themethod of claim 1, wherein the cancer comprises cancer cells thatexpress CD5.
 3. The method of claim 2, further comprising identifyingthe cancer as comprising cells that express CD5.
 4. The method of claim1, wherein the cancer is a liquid tumor.
 5. The method of claim 4,wherein the liquid tumor is a chronic lymphocytic leukemia or multiplemyeloma.
 6. The method of claim 5, wherein the chronic lymphocyticleukemia is a B cell chronic lymphocytic leukemia.
 7. The method ofclaim 1, wherein the anti-CD200 antibody, or CD200-binding fragmentthereof, inhibits the interaction between CD200 and CD200R.
 8. Themethod of claim 1, wherein the anti-CD200 antibody, or CD200-bindingfragment thereof, comprises a heavy chain CDR1 (HCDR1) comprising theamino acid sequence: GFTFSGFAMS (SEQ ID NO:4); a heavy chain CDR2(HCDR2) comprising the amino acid sequence: SISSGGTTYYLDSVKG (SEQ IDNO:5); a heavy chain CDR3 (HCDR3) comprising the amino acid sequence:GNYYSGTSYDY (SEQ ID NO:6); a light chain CDR1 (LCDR1) comprising theamino acid sequence: RASESVDSYGNSFMH (SEQ ID NO:7); a light chain CDR2(LCDR2) comprising the amino acid sequence: RASNLES (SEQ ID NO:8); and alight chain CDR3 (LCDR3) comprising the amino acid sequence: QQSNEDPRT(SEQ ID NO:9).
 9. The method of claim 1, wherein the anti-CD200antibody, or CD200-binding fragment thereof, is murine, chimeric,humanized, or fully human.
 10. The method of claim 1, wherein theCD200-binding fragment is selected from the group consisting of a Fabfragment, a F(ab′)₂ fragment, a Fab′ fragment, an scFv fragment, aminibody, a diabody, or a triabody.
 11. The method of claim 1, whereinthe cancer comprises cancer cells having reduced CD20 expression,relative to the expression of CD20 by normal cells of the samehistological type as the cells from which the cancer cells are derived.